Uma bacteriocina produzida por uma cepa de Lactobacillus sake 2a, isolada de lingüiça frescal comercial foi purificada, caracterizada e seu modo de ação foi estudado com o objetivo de ser utilizada como bioconservante em alimentos. A bacteriocina apresentou efeito bactericida contra Listeria monocytogenes em meio de cultura. Algumas cepas como Listeria welshimeri, Listeria seeligeri e Listeria inócua foram sensíveis, mas Staphylococcus aureus e Escherichia coli O157H7 foram resistentes a esta bacteriocina. O melhor meio de cultura para a sua produção foi o meio MRS à 25 ou 30°C durante a fase logarítmica de crescimento, obtendo-se 450 UA/ml de bacteriocina. Observou-se estabilidade a 121° C por 15 minutos. A bacteriocina foi purificada 71186 vezes pela técnica de adsorção/desorção de proteínas seguida de cromatografia de troca iônica Mono S, com rendimento de 3%. O peso molecular estimado foi de 3 a 6 kDa, determinado por eletroforese em gel de poliacrilamida-tricina. Nos estudos do mecanismo de ação, a bacteriocina dissipou o potencial de membrana, o gradiente de pH e reduziu de 80% os níveis de ATP intracelular não alterando os níveis de ATP extracelular. Bacteriocina 2a, nas concentrações de 28, 60 e 114 UA/ml, também causou efluxo de 3, 30 e 100% de carboxifluoresceina dos lipossomos construídos com lipídios de membrana de Listeria monocytogenes Scott A, respectivamente. Os resultados indicam que a bacteriocina 2a atua formando poros na membrana citoplasmática de células sensíveis não necessitando de um receptor específico. Conclui-se também que a bacteriocina é um bioconservante em potencial, podendo ser usada no controle de Listeria monocytogenes em alimentos. / Bacteriocin 2a produced by Lactobacillus sake 2a isolated from a Brazilian meat product (lingüiça) was purified, characterized and its mecanism of action was studied. The bacteriocin 2a showed bactericidal effect against Listeria monocytogens, Listeria welshimeri, Listeria seeligeri and Listeria inocua but it did not have an effect against Staphylococcus aureus e Escherichia coli O157H7 strains. The highest concentration of bacteriocin 2a (450 AU/ml) was found in MRS medium incubated at 25 or 30°C and its production was at its maximum towards logaritmic growth. It was stable at 121° C for 15 minutes. Bacteriocin 2a was purified 71186 fold by salt extraction from Lactobacillus sake cells, followed by cation exchange chromatography using Mono S column. It has an estimated molecular mass of 3-6kDa. In mechanistic studies of action against Listeria monocytogenes Scott A, bacteriocin 2a dissipated the pH gradient, the ΔΨ and reduced the ATP internal concentration by 80% with no detectable increase in the external ATP concentration. Bacteriocin 2a concentration of 28, 60 and 114 AU/ml, also mediated carboxyfluorescein efflux of 3, 30 and 100% from liposomes made from lipids extracted from Listeria monocytogenes Scott A, respectively. These data indicate that bacteriocin 2a forms pores in the citoplasmatic membrane of target cells similarly Class II bacteriocins. In addiction it can be use as a potential anti-microbial against Listeria monocytogenes in food.

Triclosan (TCS) is a personal care product with many fields of application and is of public interest for several years now. Monitoring studies showed that TCS is a ubiquitous chemical in the aquatic environment. Aquatic organisms are exposed to TCS in a broad range of concentrations, from ng L-1 up to lower μg L-1. TCS has a bactericidal effect for various types of gram-positive and gram negative bacteria. TCS targets a specific bacterial fatty acid biosynthetic enzyme, enoyl-[acyl-carrier protein] reductase (Schweizer, 2001). Therefore the terminal reaction in the fatty acid elongation cycle is inhibited (Levy et al., 1999). Although effects on non-target organisms are reported, the Mode of Action (MoA) of TCS is not well examined for those organisms. The aim of this PhD thesis was to investigate effects of TCS on non-target autotrophic organisms at different levels of biological complexity in the aquatic environment. In this thesis microalgae have been found to be very sensitive to TCS. In some cases even higher sensitivities than in bacteria were observed, which is in accordance with published effect data (Harada et al., 2008; Orvos et al., 2002). Similarly to bacteria, high species sensitivity differences were observed for algae (Franz et al., 2008). In bacteria these sensitivity differences can be ascribed to several resistance mechanisms reported in Schweizer (2001). These findings lead to the question about the reasons for species sensitivity differences in algae. A mesocosm study was performed to detect effects of TCS across levels of biological organization and to investigate the impact of sensitivity differences on complex aquatic communities. For that purpose, structural and functional effects parameters were observed.

<p>  </p> <p>Dicamba (3,6-dichloro-2-methoxybenzoic acid) and 2,4-D (2,4-dichlorophenoxyacetic acid) are two widely used herbicides for broadleaf weed control in soybeans. However, off-target application of dicamba and 2,4-D can cause severe damage to sensitive vegetation and crops. Early detection and assessment of off-target damage caused by these herbicides are necessary to help plant diagnostic labs and state regulatory agencies collect more information of the on-site conditions so to develop solutions to resolve the issue in the future. In 2021, the study was conducted to detect damage to soybean leaves caused by dicamba and 2,4-D by using LeafSpec, an accurate handheld hyperspectral leaf scanner. . High resolution single leaf hyperspectral images of 180 soybean plants in the greenhouse exposed to nine different herbicide treatments were taken 1, 7, 14, 21 and 28 days after herbicide spraying. Pairwise PLS-DA models based on spectral features were able to distinguish leaf damage caused by two different modes of action herbicides, specifically dicamba and 2,4-D, as early as 2 hours after herbicide spraying. In the spatial distribution analysis, texture and morphological features were selected for separating the dosages of herbicide treatments. Compared to the mean spectrum method, new models built upon the spectrum, texture, and morphological features, improved the overall accuracy to over 70% for all evaluation dates. The combined features are able to classify the correct dosage of the right herbicide as early as 7 days after herbicide sprays. Overall, this work has demonstrated the potential of using spectral and spatial features of LeafSpec hyperspectral images for early and accurate detection of dicamba and 2,4-D damage in soybean plants.</p> <p>   </p>

Worldwide, malaria is one of the deadliest diseases. In 2016 it sickened 216 million people and caused 445,000 deaths. In order to control the spread of this deadly diseases to human, we can either target the mosquito vector (Anopheles gambiae) or the parasite (Plasmodium falciparum). Due to recent emergence of resistance to current insecticides and antimalarial drugs there is a pressing need to discover and develop new agents that engage new targets in these organisms. To circumvent the effect of resistance to pyrethroid insecticides on the efficacy of insecticide treated nets (ITNs), the use of acetylcholinesterase (AChE) inhibitors on ITNs has drawn attention. In the first project, we explored a small library of γ- substituted oxoisoxazole- 2(3H)-carboxamides and isoxazol-3-yl carbamates, and nitriles as AChE inhibitors targeting wild- type (G3) and resistant (Akron) An. gambiae mosquito. In total 23 compounds were synthesized and evaluated. Both carbamates and carboximides with a 2-cyclopropylethyl side chain (1-87a and 1-88a) were extremely toxic to Akron mosquitos, yet these compounds did not exhibit appreciable selectivity between human and An. gambiae AChE. Unfortunately, none of the nitriles showed appreciable toxicity to G3 strain of the mosquitoes, nor did they inhibit An. gambiae AChE. In the second project, conducted in collaboration with Professor Michael Klemba, we focused on the mode of action of an established antimalarial drug, Mefloquine (MQ). Dr. Klemba's recently developed amino acid efflux assay was used to determine the effect of MQ and its open-ring analogs on hemoglobin endocytosis and catabolism in P. falciparum-infected erythrocytes. In total 26 MQ analogs were synthesized and 18 were studied in depth to determine their potency to inhibit leucine (Leu) efflux and parasite growth (SYBR Green). An excellent correlation (R² = 0.98) over nearly 4 log units was seen for these 18 compounds in the two assays. These data are consistent with the hypothesis that the antimalarial action of these compounds principally derives from inhibition of hemoglobin endocytosis. After this observation, a number of photo-affinity probes were designed and synthesized in hopes of isolating the molecular target of MQ. These analogs are currently being used by Dr. Klemba in pull-down experiments. In the third project, conducted in collaboration with Professor Belen Cassera, we sought to optimize a new antimalarial drug lead that would circumvent current resistance mechanisms. In Plasmodium parasites, the methylerythritol phosphate (MEP) pathway is known to be essential for its growth. This pathway is absent in humans, presenting the opportunity to develop potentially safe and effective therapeutic candidates. Previous work in the Cassera and Carlier lab had established that MMV008138 was the only compound in the Malaria Box that targeted the MEP pathway and that it was (1R,3S)-configured. My research expanded previous efforts in the Carlier group and produced synthesis of 73 analogs of MMV008138 (3-21a'1) that were tested for growth inhibition. These analogs featured variation at the A-, B-, C- and D-ring. In the process, a novel Pictet-Spengler ring expansion reaction of ortho-substituted acetphenones was discovered. The ring-expanded products were identified by means of 1D and 2D NMR experiments, HRMS, and X-ray crystallography. Among the 73 analogs prepared, four compounds showed similar growth inhibition potency to the lead 3-21a'1. In particular, the methoxyamide 3-80a, and the fluorinated A-ring analogs 3-124a, 3-124c and 3-124d all showed excellent (500-700 nM) growth IC₅₀ values against P. falciparum. All four showed full rescue upon co-application of IPP (200 μM), confirming that they target the MEP pathway. ADME-Tox evaluation of these new analogs will soon be underway. / PHD / Malaria is a severe and potentially fatal mosquito-borne disease. The continuous emergence of insecticide-resistant mosquitoes and drug-resistant parasite strains necessitates the development of novel antimalarial agents, notably those that engage new targets in these organisms. Herein we present three projects in which the synthesis and characterization of new malaria insecticide and therapeutic candidates are described. Our aim in the first project was to synthesize acetylcholinesterase (AChE) inhibitors as potential mosquitocides to be deployed on insecticide-treated nets. Three different classes of compounds were synthesized and characterized. Their potency to inhibit the wild-type and insecticide-resistant mosquito AChE, and their corresponding mosquito toxicities were assessed. Mosquito-toxic compounds were identified, but they did not show appreciable selectivity between mosquito and human AChEs. The second project was directed toward finding the biological target of a known antimalarial drug; mefloquine (MQ). Numerous different MQ analogs were synthesized, and their potency was assessed in two biochemical assays. The results of this study strongly suggest that MQ kills malaria parasites by preventing them from ingesting the red blood cell hemoglobin. The third project was concerned with the optimization of a compound (MMV008138) that kills malaria parasites by preventing it from synthesizing a key biochemical building block (IPP). Several new compounds were prepared that had similar antimalarial activity to MMV008138, of which many have better potential to serve as antimalarial drugs. In addition, these studies provided valuable insights for the design of further improved analogs.

L’existence d’un « régulateur financier » ayant pour mission d’encadrer les marchés financiers est un phénomène répandu qui confronte les systèmes juridiques à une question délicate : celle de l’intégration de ce régulateur dans le paysage institutionnel classique. C’est cette question qui est au coeur de la recherche menée en droit comparé, en France et au Moyen-Orient. D’origine anglo-saxonne, la formule frappe par son originalité fonctionnelle et structurelle. L'appréciation du phénomène passe d'abord par l’étude de la spécificité de la fonction de régulation. Comment appréhender le fait que le régulateur financier cumule des compétences normatives, contentieuses et administratives ? Ne dessaisit -il pas le législateur, le juge et l’exécutif d'une partie de leur activité? L’analyse révèle que la raison d'être du régulateur financier est de fonctionner en complémentarité avec les pouvoirs de l’État. Ne constituant pas un quatrième pouvoir, le régulateur financier diffuse l’art de la régulation résultant de son statut de gendarme de la Bourse et de magistère moral. L’approche fonctionnelle est complétée par une analyse du statut du régulateur financier. Quel positionnement occupe-t-il, alors qu'il combine des éléments privés et publics ? L’étude montre que le régulateur financier résiste aux distinctions classiques. Dépassant les frontières public/privé, il est à mi-chemin entre l'Etat et le marché. Il se nourrit des valeurs du privé par l’association des professionnels dans la régulation, le recours à des mécanismes contractuels pour régler les différends et la soumission au contrôle du juge judiciaire. Mais il maintient en même temps un statut public spécifique en vue d’assurer son indépendance. Il en résulte l'émergence d'un nouveau mode d’action de troisième voie ayant pour objet la mise en oeuvre d'une nouvelle fonction de l'Etat qui est la régulation dont l'avènement nécessite la conception d'une formule institutionnelle inédite. / The existence of a "financial regulator" whose mission is to control the financial markets is a widespread phenomenon that faces legal systems with a delicate issue: that of the integration of this regulator in the classic institutional landscape. It is this question which is at the core of research in comparative law between France and the Middle East. The formula, being of an Anglo-Saxon origin, is intriguing for its functional and structural originality. The evaluation of this phenomenon begins with the study of the specificity of the regulatory function. The question is how to apprehend that the financial regulator combines normative, administrative and litigation functions. Would not it divest the legislator, the judge and the executive of a part of their own activities? The analysis reveals that the purpose of the financial regulator is to function as a complement to the State’s powers. The financial regulator doesn’t constitute a fourth power itself; it rather diffuses the art of the regulation resulting from its status as a markets watchdog and its moral authority. The functional approach is complemented by examining the status of the financial regulator. What position does it occupy while combining both private and public elements? The study shows that the financial regulator is resistant to conventional legal distinctions. It is halfway between the State and the market surpassing by that the boundaries of the public/private law. In fact, it is nourished by private values through associating professionals in the regulation, using contractual mechanisms to resolve disputes, and submission to the judicial court control. However, it maintains, at the same time, a specific public status to ensure its independence. The result is the emergence of a new third mode of action whose purpose is the exercise of a new function of the State, which is the regulation, whose advent requires designing an unprecedented institutional formula.

Matrix remodeling could be an important mode of action of multipotent mesenchymal stromal cells (MSC) in extracellular matrix (ECM) disease, but knowledge is limited in this respect. As MSC are well-known to adapt their behavior to their environment, we aimed to investigate if their mode of action would change in response to healthy versus pathologically altered ECM. Human MSC-derived ECM was produced under different culture conditions, including standard culture, culture on Matrigel-coated dishes, and stimulation with the pro-fibrotic transforming growth factor-1 (TGF1). The MSC-ECM was decellularized, characterized by histochemistry, and used as MSC culture substrate reflecting different ECM conditions. MSC were cultured on the different ECM substrates or in control conditions for 2 days. Culture on ECM increased the presence of surface molecules with ECM receptor function in the MSC, demonstrating an interaction between MSC and ECM. In MSC cultured on Matrigel-ECM and TGF1-ECM, which displayed a fibrosis-like morphology, gene expression of collagens and decorin, as well as total matrix metalloproteinase (MMP) activity in the supernatant were decreased as compared with control conditions. These results demonstrated that MSC adapt to their ECM environment, which may include pathological adaptations that could compromise therapeutic efficacy.

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.

Target-based screening projects for bioactive (orphan) compounds have been shown in many cases to be insufficiently predictive for in vivo efficacy, leading to attrition in clinical trials. Phenotypic screening has hence undergone a renaissance in both academia and in the pharmaceutical industry, partly due to this reason. One key shortcoming of this paradigm shift is that the protein targets modulated need to be elucidated subsequently, which is often a costly and time-consuming procedure. In this work, we have explored both improved methods and real-world case studies of how computational methods can help in target elucidation of phenotypic screens. One limitation of previous methods has been the ability to assess the applicability domain of the models, that is, when the assumptions made by a model are fulfilled and which input chemicals are reliably appropriate for the models. Hence, a major focus of this work was to explore methods for calibration of machine learning algorithms using Platt Scaling, Isotonic Regression Scaling and Venn-Abers Predictors, since the probabilities from well calibrated classifiers can be interpreted at a confidence level and predictions specified at an acceptable error rate. Additionally, many current protocols only offer probabilities for affinity, thus another key area for development was to expand the target prediction models with functional prediction (activation or inhibition). This extra level of annotation is important since the activation or inhibition of a target may positively or negatively impact the phenotypic response in a biological system. Furthermore, many existing methods do not utilize the wealth of bioactivity information held for orthologue species. We therefore also focused on an in-depth analysis of orthologue bioactivity data and its relevance and applicability towards expanding compound and target bioactivity space for predictive studies. The realized protocol was trained with 13,918,879 compound-target pairs and comprises 1,651 targets, which has been made available for public use at GitHub. Consequently, the methodology was applied to aid with the target deconvolution of AstraZeneca phenotypic readouts, in particular for the rationalization of cytotoxicity and cytostaticity in the High-Throughput Screening (HTS) collection. Results from this work highlighted which targets are frequently linked to the cytotoxicity and cytostaticity of chemical structures, and provided insight into which compounds to select or remove from the collection for future screening projects. Overall, this project has furthered the field of in silico target deconvolution, by improving the performance and applicability of current protocols and by rationalizing cytotoxicity, which has been shown to influence attrition in clinical trials.

El moteado del níspero, causado por el hongo Fusicladium eriobotryae es la principal enfermedad que afecta al cultivo del níspero, produciendo pérdidas importantes en la cosecha en los años con condiciones climáticas adecuadas. Sin embargo, no existen estudios sobre la epidemiología y el control de esta enfermedad, por lo que éstos constituyen el principal objetivo de la presente Tesis. Para ello, se va ha caracterizar in vitro y en campo la influencia de las principales variables climáticas en el desarrollo de F. eriobotryae, desarrollándose ecuaciones matemáticas que modelicen esta relación. Por otro lado, se va a llevar a cabo un modelo epidemiológico para el moteado del níspero capaz de predecir, en función de las principales variables climáticas el riesgo de infección. Además, se realizará un estudio del control de la enfermedad que comprenderá, por un lado, la evaluación in vitro y en planta de la efectividad de las principales materias activas utilizadas para el control del moteado del níspero, y por otro la evaluación del grado de resistencia de una colección de aislados de F. eriobotryae a DMI y Metil-Tiofanato y la caracterización molecular de la misma. / González Domínguez, E. (2014). Biology, epidemiology and control of Fusicladium eriobotryae, causal agent of loquat scab [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/38715