No description available.

<p>Cadmium (Cd) is a highly mobile and toxic heavy metal that negatively affects plants, soil biota, animals and humans, even in very low concentrations. Currently, Cd contamination of cocoa produced in Latin American countries is a significant problem, as concentrations can exceed acceptable levels set by the European Union (0.5 mg/kg), sometimes by more than 10 times allowable levels. In South America, <i>Theobroma cacao</i> is an essential component of the basic market basket. This crop contributes to the Latin-American trade balance, as these countries export cacao and chocolate-based products to major consumer countries such as the United States and Europe. Some soil amendments can alter the bioavailability and uptake of Cd into edible plant tissues, though cacao plants can accumulate Cd without displaying any visible symptoms of phytotoxicity, which makes it difficult to determine if potential remediation strategies are successful. Currently, the only effective way to quantify Cd accumulation in plant tissues is via destructive post-harvest practices that are time-consuming and expensive. New hyperspectral imaging (HSI) technologies developed for use in high-throughput plant phenotyping are powerful tools for monitoring environmental stress and predicting the nutritional status in plants. Consequently, the experiments described in this thesis were conducted to determine if HSI technologies could be adapted for monitoring plant stress caused by Cd, and estimating its concentration in vegetative plant tissues. Two leafy green crops were used in these experiments, basil (<i>Ocimum basilicum L.</i> var. Genovese) and kale (<i>Brassica oleracea L</i>. var. Lacinato), because they are fast growing, and therefore, could serve as indicator crops on cacao farms. In addition, we expected these two leafy green crops would differ in their morphological responses to Cd stress. Specifically, we predicted that stress responses would be visible in basil, but not kale, which is known to be a hyperaccumulator. The plants were subject to four levels of soil Cd (0, 5, 10 and 15 ppm), and half of the pots were amended with biochar at a rate of 3% (v/v), as this amendment has previously been demonstrated to improve plant health and reduce Cd uptake. The experiments were conducted at Purdue’s new Controlled Environment Phenotyping Center (CEPF). The plants were imaged weekly and manual measurements of plant growth and development were collected at the same times, and concentrations of Cd as well as many other elements were determined after harvest. Fourteen vegetation indices generated using HSI images collected from the side and top view of plants were evaluated for their potential to identify subtle signs of plant stress due soil Cd and the biochar amendment. In addition, three mathematical models were evaluated for their potential to estimate Cd concentrations in the plant biomass and determine if they exceed safe standards (0.28 mg/kg) set by the Food and Agriculture Organization (FAO) for leafy greens. Results of these studies confirm that like many plants, these leafy green crops can accumulate Cd levels that are well above safety thresholds for human health, but exhibit few visible symptoms of stress. The normalized difference vegetation index (NDVI) and the chlorophyll index at the red edge (CI_RE) were the best indices for detecting Cd stress in these crops, and the plant senescence and reflectance index (PSRI) and anthocyanin reflectance index (ARI) were the best at detecting subtle changes in plant physiology due to the biochar amendment. The heavy metal stress index (HMSSI), developed exclusively for detecting heavy metal stress, was only able to detect Cd stress in basil when images were taken from the top view. Results of the mathematical models indicated that principal components analysis (PCA) and partial least squares (PLS) models overfit despite efforts to transform the data, indicating that they are not capable of predicting Cd concentrations in these crops at these levels. However, the artificial neural networks (ANN) was able to predict whether leafy greens had levels of Cd that were above or below critical thresholds suggested by the FAO, indicating that HSI could be further developed to predict Cd concentrations in plant tissues. Further research conducted in the field and in the presence of other environmental stress factors are needed to confirm the utility of these tools, and determine whether they can be adapted to monitor Cd uptake in cacao plants.</p>

No description available.

Maintaining cell envelope integrity is of vital importance for all microorganisms. Not surprisingly, evolution has shaped conserved protein protection networks that connect stress perception, transmembrane signal transduction, and mediation of cellular responses upon cell envelope stress. The phage shock protein (Psp) stress response is one such conserved protection network. Most knowledge about the Psp response derives from studies in the Gram-negative model bacterium Escherichia coli, where the Psp system consists of several well-defined protein components. Homologous systems were identified in representatives of the Proteobacteria, Actinobacteria, and Firmicutes. However, the Psp system distribution in the microbial world remains largely unknown. By carrying out a large-scale, unbiased comparative genomics analysis, we found components of the Psp system in many bacterial and archaeal phyla and describe that the predicted Psp systems deviate dramatically from the known prototypes. The core proteins PspA and PspC have been integrated into various (often phylum-specifically) conserved protein networks during evolution. Based on protein domain-based and gene neighborhood analyses of pspA and pspC homologs, we built a natural classification system for Psp networks in bacteria and archaea. We validate our approach by performing a comprehensive in vivo protein interaction study of Psp domains identified in the Gram-positive model organism Bacillus subtilis and found a strong interconnected protein network. Our study highlights the diversity of Psp domain organizations and potentially diverse functions across the plethora of the microbial landscape, thus laying the ground for studies beyond known Psp functions in underrepresented organisms.

Les gènes de l'isocitrate déshydrogénase (IDH) sont mutés dans 70 à 80 % des gliomes de bas grade. Les enzymes mutantes IDH qui en résultent présentent une activité de gain de fonction, produisant du R-2-hydroxyglutarate (R-2-HG), appelé oncométabolite en raison de son accumulation anormale dans les tumeurs et de ses activités oncogéniques potentielles. Parmi les caractéristiques du cancer telles que la reprogrammation métabolique et épigénétique, le stress réplicatif et la stabilité du génome ont été peu caractérisés dans les cancers IDH-mutants. Par conséquent, cette étude vise à étudier l'impact de l'accumulation de R-2-HG sur la réplication de l'ADN et sa contribution au stress réplicatif dans les cancers IDH-mutants. Nous avons étudié la dynamique de la fourche de réplication dans des astrocytes humains normaux et confirmé les résultats dans d'autres lignées cellulaires normales et cancéreuses. Nous avons constaté que le traitement exogène par l'octyl-R-2-HG entravait la progression de la fourche de réplication et retardait par conséquent l'achèvement de la phase S. L'évaluation des niveaux de phosphorylation des protéines RPA, CHK1 et H2AX a révélé que la réponse classique au stress réplicatif (RSR) n'était pas activée. Un état cellulaire dans lequel la réplication de l'ADN est altérée sans activation de la RSR a notamment été décrit dans la littérature comme résultant de l'activation de la réponse au stress intégré (ISR). Cependant, l'activation de la RSI dans les cancers mutants IDH n'est pas bien étudiée. En évaluant les marqueurs d'activation de la RSI, tels que la phosphorylation de l'eIF2α et les niveaux de protéines ATF4, nous avons montré que l'octyl-R-2-HG activait la RSI. De plus, le blocage de l'ISR a partiellement sauvé la fourche de réplication et la progression de la phase S. Nous avons répliqué cette étude oncométrique. Nous avons reproduit ce défaut de réplication de l'ADN lié à l'oncométabolite ainsi que l'effet de sauvetage partiel de l'ISRIB lors de l'induction de la surexpression du gène IDH mutant. Nos résultats indiquent que la production de R-2-HG associée à la mIDH peut inhiber la dynamique normale de réplication de l'ADN via la signalisation ISR. / The isocitrate dehydrogenase (IDH) genes are mutated in 70-80% of low-grade gliomas. The resulting IDH mutant enzymes exhibit gain-of-function activity, producing R-2-hydroxyglutarate (R-2-HG), which is referred to as an oncometabolite due to its abnormal accumulation in tumours and potential oncogenic activities. Among the hallmarks of cancer such as metabolic and epigenetic reprogramming, replicative stress and genome stability have been poorly characterized in IDH-mutant cancer. Therefore, this study aims to investigate the impact of R-2-HG accumulation on DNA replication and its contribution to replicative stress in IDH-mutant cancers. We investigated replication fork dynamics in normal human astrocytes and confirmed the results in other normal and cancer cell lines. We found that exogenous treatment with octyl-R-2-HG impaired replication fork progression and consequently delayed S-phase completion. Assessment of RPA, CHK1 and H2AX protein phosphorylation levels revealed that the classical Replicative Stress Response (RSR) was not activated. Among others, a cell state in which DNA replication was impaired without activation of the RSR has been described in the literature as a result of activation of the Integrated Stress Response (ISR). However, ISR activation in IDH-mutant cancers is not well studied. Hence, by assessing ISR activation markers such as eIF2α phosphorylation and ATF4 protein levels, we showed that octyl-R-2-HG activated ISR. Moreover, blocking ISR partially rescued the replication fork and S-phase progression. We replicated this oncometabolite-related DNA replication defect as well as ISRIB’s partial rescue effect upon induction of mutant IDH gene overexpression. Our results indicate that mIDH-associated R-2-HG production possibly inhibits normal DNA replication dynamics via ISR signalling.

Bakterielle Wirte wie Escherichia coli dienen der Produktion industrieller rekombinanter Proteine. Dieser Prozess verursacht systemischen Stress und führt zu umfangreichen Veränderungen in mRNA- und Proteinexpression. In meiner Arbeit analysiere ich Regulationsmechanismen der zellulären Reaktion auf diesen Stress. Zudem untersuche ich die zelluläre Ressourcenallokation mittels eines stationären Ganzzellmodells von E. coli, basierend auf der Resource Balance Analysis. Das Modell berücksichtigt Kosten zellulärer Prozesse und Einschränkungen wie Energie, Effizienz und Raum. Es unterstützt die Experimentplanung in der Bioproduktion. Weiterhin habe ich an der Entwicklung von RBApy mitgewirkt, einer Software zur Erstellung und Simulation von RBA-Modellen. Schließlich entwickle ich ein Modell zur Untersuchung der Regulation von Stressreaktionen durch die Tendenz der Zelle, wachstumsoptimale Ressourcenstrategien anzuwenden. Das Modell berücksichtigt zelluläre Beschränkungen und zeigt, dass die erhaltene Stressreaktion der experimentell ermittelten Reaktion ähnelt. Die Integration von Ressourcenzuteilung in Zellmodelle ermöglicht Einsichten in regulatorische Ereignisse und Anpassungen während der Bioproduktion, was zur Optimierung der rekombinanten Proteinexpression in Escherichia coli beiträgt. / Bacterial hosts such as Escherichia coli are used for the production of industrial recombinant proteins. This process causes systemic stress and leads to extensive changes in mRNA and protein expression. In my work, I analyze regulatory mechanisms of the cellular response to this stress. In addition, I investigate cellular resource allocation using a steady-state whole-cell model of E. coli based on resource balance analysis. The model accounts for costs of cellular processes and constraints such as energy, efficiency, and space. It supports experiment design in bioproduction. Furthermore, I contributed to the development of RBApy, a software to create and simulate RBA models. Finally, I developed a model to study the regulation of stress responses by the tendency of the cell to adopt growth-optimal resource strategies. The model accounts for cellular constraints and shows that the obtained stress response resembles the experimentally determined response. Integrating resource allocation into cell models provides insights into regulatory events and adaptations during bioproduction, which contributes to the optimization of recombinant protein expression in Escherichia coli.

No description available.

No description available.

Over the course of the last decades, the continuous exposure of bacteria to antibiotics—at least in parts due to misprescription, misuse, and misdosing—has led to the widespread development of antimicrobial resistances. This development poses a threat to the available medication in losing their effectiveness in treating bacterial infections. On the drug development side, only minor advances have been made to bring forward novel therapeutics. In addition to increasing the efforts and approaches of tapping the natural sources of new antibiotics, synthetic approaches to developing novel antimicrobials are being pursued. In this study, BDTL049 was rationally designed using knowledge based on the properties of natural antibiotics. BDTL049 is a carbosilane dendritic system with bow-tie type topology, which has antimicrobial activity at concentrations comparable to clinically established natural antibiotics. In this report, we describe its mechanism of action on the Gram-positive model organism Bacillus subtilis. Exposure to BDTL049 resulted in a complex transcriptional response, which pointed toward disturbance of the cell envelope homeostasis accompanied by disruption of other central cellular processes of bacterial metabolism as the primary targets of BDTL049 treatment. By applying a combination of whole-cell biosensors, molecular staining, and voltage sensitive dyes, we demonstrate that the mode of action of BDTL049 comprises membrane depolarization concomitant with pore formation. As a result, this new molecule kills Gram-positive bacteria within minutes. Since BDTL049 attacks bacterial cells at different targets simultaneously, this might decrease the chances for the development of bacterial resistances, thereby making it a promising candidate for a future antimicrobial agent.

No description available.