More than a decade after the completion of the human genome project, the function of a large number of genes remains to be elucidated. Forward and reverse genetic approaches have proven to be powerful tools to study gene function and have provided insights into fundamental biological processes. Furthermore, functional genetic screening can lead to a better understanding of the action of endogenous and exogenous stimuli such as hormones or drugs on biological systems. Thus far, systematic and unbiased studies have largely been limited to model organisms. However, complex disease-relevant genotypes and phenotypes cannot be studied in entirety in lower organisms creating a need for systematic approaches in human cells. This thesis describes a series of studies using forward and reverse genetic approaches combined with state-of-the-art technology in haploid human cells. The first chapter describes the development of a quantitative phenotypic read-out using a novel application of RNA-sequencing that allows the functional annotation of genes in signalling pathways. The presented data demonstrate that the employed shallow RNA-sequencing method is scalable and suitable as a read-out for reverse genetic screening. The second chapter focuses on the implementation of this method in a large reverse genetic study in human cells to functionally annotate tyrosine kinases in signalling pathways upon stimulation with a set of ten polypeptides and small molecules. The screens revealed known and unexpected interactions between different signalling molecules and pathways, validating the technical approach in a biological context. The third chapter presents a pilot study describing the set-up of a forward genetic technique for compound mode-of-action screening using a pooled human mutant cell line collection. The chemical genetic approach displayed sufficient sensitivity and allowed to monitor thousands of gene-drug interactions simultaneously. Together, this thesis combines elements to advance technological and biological aspects of functional genomics and chemical genetics.

Les accélérateurs sont des adjuvants couramment employés dans les matériaux cimentaires afin d’accélérer leur prise et/ou leur durcissement. Leur ajout permet d’accélérer les cadences de production de béton préfabriqué ou encore de compenser le retard d’hydratation induit par l’emploi d’ajouts minéraux cimentaires ou par des conditions hivernales. De nombreuses espèces chimiques, molécules ou ions, connues pour leur pouvoir accélérateur ont été étudiées dans la littérature. Néanmoins, souvent leur mode d’action est associé à une simple activation chimique, se caractérisant par l’accélération de l’hydratation de certaines phases cimentaires. Or au sein de la pâte cimentaire, ces adjuvants peuvent aussi avoir une action physique et modifier l’état de dispersion des grains de ciment, ce qui pourrait indirectement influencer la réactivité chimique.L’objectif de ce travail est donc d’étudier les modes d’action de différents systèmes accélérateurs en combinant l’analyse de la réactivité chimique du ciment et de l’organisation mésostructurale. Plusieurs amines de structures moléculaires différentes et plusieurs sels sodiques ont été employés seuls ou de manière combinée dans des pâtes cimentaires et des mortiers normalisés. Ces matériaux ont alors été étudiés de l’échelle nano/microscopique, puis mésoscopique à macroscopique, sur une période de temps s’étalant des premières minutes d’hydratation jusqu’à 28 jours. L’analyse du liquide interstitiel des pâtes cimentaires a permis d’évaluer les capacités de sorption et de complexation des adjuvants. Via des suivis d’hydratation par calorimétrie isotherme, diffraction des rayons X et analyse thermogravimétrique, l’impact des accélérateurs sur la dissolution des phases cimentaires anhydres et la précipitation d’hydrates a été déterminé. En parallèle, l’organisation mésostructurale des suspensions cimentaires adjuvantées a été analysée de manière directe par microscopies et granulométrie laser. Le comportement rhéologique et le suivi de sédimentation ont aussi permis d’appréhender indirectement l’état de dispersion des pâtes cimentaires. Pour finir, à l’échelle du mortier, des mesures de résistance à la compression ont été réalisées et la microstructure des échantillons a été étudiée par porosimétrie mercure et surface spécifique BET.La combinaison de ces caractérisations chimiques, physico-chimiques et granulaires a permis de mettre en évidence les principaux modes d’action des différentes familles d’accélérateurs. En particulier l’influence sur l’accélération de la structure moléculaire des amines et des espèces ioniques présentes dans le sel a été appréhendée. Enfin, l’emploi de combinaisons d’accélérateurs a permis de moduler l’accélération d’hydratation du matériau cimentaire et d’obtenir des effets synergiques. / Accelerators are common cement additives used in order to accelerate setting and/or hardening. Their addition allows accelerating production rates of precast concrete or compensating hydration retardation induced by the use of supplementary cementitious materials or by winter conditions. Several chemical species, molecules or ions, known for their accelerator power have been studied in the literature. However, their mode of action is often simply associated to chemical activation, which is characterized by hydration acceleration of particular cement phases. Yet, inside the cement paste, these additives can also have a physical action and modify the dispersion state of cement grains, which might indirectly influence chemical reactivity.Therefore the objective of this work is to study the modes of action of different accelerator systems by combining the analysis of cement chemical reactivity and mesostructural organization. Some amines with different molecular structures and some sodic salts were used individually or in a combined way in cement pastes and normalized mortars. These materials were then studied from nano/microscopic to mesoscopic and macroscopic scale over a period of time ranging from the first minutes of hydration till 28 days. The analysis of the pore solution allowed evaluating the additive sorption and complexation ability. Cement hydration was followed by isothermal calorimetry, X-ray diffraction and thermogravimetric analysis in order to determine the impact of accelerators on anhydrous cement phase dissolution and hydrate precipitation. In parallel, mesostructural organization of admixtured cement suspensions was analyzed indirectly by microscopy and laser granulometry. Rheological and sedimentation behavior also allowed apprehending indirectly the dispersion state of cement paste. Lastly, at mortar scale, compressive strength measurements were performed and sample microstructure was studied by mercury intrusion porosimetry and BET specific surface.The combination of these chemical, physicochemical and granular characterizations allowed highlighting the main modes of action of the different accelerator systems. In particular, the influence on acceleration of amine molecular structure and salt ionic species was apprehended. Finally, the use of combinations of accelerators allowed modulating the hydration acceleration of cementitious materials and obtaining synergetic effects.

A citricultura brasileira ocupa lugar de destaque no agronegócio nacional, sendo o país o maior produtor de laranja e suco de laranja do mundo. A faia (Fagus sylvatica) é uma das principais espécies das florestas na Europa, sendo usada como ornamental e por possui alto valor econômico devido à produção de madeira. Um dos principais entraves no cultivo dessas espécies é a ocorrência de doenças principalmente as causadas por espécies de Phytophthora que além de causar grandes prejuízos, os métodos de controle são difíceis e onerosos. Existem trabalhos na literatura que apontam os fosfitos como alternativa sustentável, eficaz e economicamente viável para o controle de doenças causadas por oomicetos. Entretanto, o Comitê de Ação a Resistência à Fungicidas (FRAC) classifica os fosfitos, como produtos com ingrediente ativo sem mecanismo de ação definido. Neste contexto, este trabalho teve como objetivo avaliar o produto comercial à base de fosfito de potássio, Phytogard® no controle das doenças causadas por Phytophthora nicotianae em citros e Phytophthora plurivora em faia, bem como avaliar através de análises bioquímicas se esse produto induz resistência em plântulas de citros. Além disso, foram realizados estudos in vitro para avaliar o efeito direto do Phytogard® sobre o desenvolvimento de P. nicotianae e P. plurivora e verificar os possíveis mecanismos de ação desse produto sobre esses patógenos. Foram utilizadas plântulas de citros e faia que foram aspergidas com diferentes concentrações de Phytogard® e, posteriormente, inoculadas com os patógenos. Foram avaliadas a incidência das doenças, o consumo de água e a quantidade de DNA dos patógenos nos tecidos das raízes dos hospedeiros. Ao final do experimento, foram realizadas análises bioquímicas dos tecidos das plântulas de citros. Nos experimentos in vitro, o micélio dos patógenos foi exposto a concentrações crescentes de Phytogard® sendo determinado o crescimento micelial, produção de massa fresca de micélio e de zoósporos. Avaliou-se a perda de eletrólitos, peroxidação de lipídios e a atividade da enzima β-1,3 glucanase do micélio exposto ao produto. Foi avaliada também a morfologia das hifas tratadas ou não com o Phytogard® através da técnica de Microscopia Eletrônica de Varredura (MEV). Os resultados mostraram que o Phytogard®, em todas as concentrações aplicadas controla de maneira preventiva as doenças causadas por P. nicotianae e P. plurivora em citros e faia, respectivamente. Em citros, o produto altera a atividade de algumas proteínas relacionadas à patogênese, mas não é possível concluir que o controle seja mediado por indução de resistência. O Phytogard® inibe o crescimento micelial e a produção de zoósporos de P. nicotianae e P. plurivora. Além disso, o produto modifica a morfologia das hifas, atua na permeabilidade de membrana e na síntese de parede celular do micélio dos patógenos. / The Brazilian citrus production is important to national agribusiness, and the country is the largest orange and orange juice world producer. Beech (Fagus sylvatica) is one of the main forest species in Europe and is used as ornamental and also it has a high economic value due to the production of wood. One of the main problems in the cultivation of these species is the occurrence of diseases, mainly caused by Phytophthora species. Besides causing extensive damage control methods are difficult and costly. There are studies in the literature that show phosphites as a sustainable alternative, effective and economically viable for the control of diseases caused by oomycetes. However, the Fungicide Resistance Action Committee (FRAC), classifies the phosphites as products with the active ingredient with no defined mode of action. In this context, this work aimed to evaluate the commercial product based on potassium phosphite Phytogard® on the control of diseases caused by Phytophthora nicotianae in citrus plants and Phytophthora plurivora in beech plants and evaluate through biochemical assays whether this product induces resistance in citrus seedlings. In addition, in vitro studies were carried out to evaluate the direct effect of Phytogard® on the development of P. nicotianae and P. plurivora and to understand the possible mode of action of the product on these pathogens. Citrus seedlings were sprayed with different concentrations of Phytogard® and then inoculated with the pathogen. We evaluated the diseases incidence, water uptake and the amount of pathogens DNA in the host roots. At the end of the experiment, biochemical assays with citrus seedlings were made. In in vitro experiments, the pathogen mycelium was exposed to increasing concentrations of Phytogard® and mycelial growth, production of fresh mycelium and zoospores by these pathogens were determined. We also evaluated the electrolyte leakage, lipid peroxidation and the β-1,3 glucanase activity in the mycelium exposed to the product. It was also evaluated the hyphae morphology from mycelium treated or not with Phytogard® by using scanning electron microscopy. The results showed that in all concentrations of Phytogard® preventively controled diseases caused by P. nicotianae and P. plurivora in citrus and beech plants, respectively. In citrus, the product changed the activities of some pathogenesis-related proteins, but it is not possible to conclude that the control was mediated by resistance induction. The Phytogard® inhibited mycelial growth and zoospore production by P. nicotianae and P. plurivora. Furthermore, the product modified the hyphae morphology, changed membrane permeability and mycelium cell wall synthesis in the pathogens.

The need for profitable soybean production practices gain continually with increasing input costs and reduced profit margins. Constant cultivar and product developments has resulted in limited current data available regarding the profitability of preventative fungicide applications and physiological benefits that can occur from these applications. Research was conducted during 2017 and 2018 to determine optimal fungicide application timing, while assessing multiple fungicide options and resulting effect on soybean grain yield, seed quality, and profitability. Additional research was conducted to determine optimal row spacing, planting date and fungicide application combinations to maximize soybean production profitability. These data suggest using multi-mode of action fungicide treatments increased soybean grain yield, regardless of application timing. These data also suggest, profitability from application of multi-mode of action fungicides can be observed at lower adjusted market prices.

Drimane and coloratane sesquiterpenoids are present in several plants, microorganisms, and marine life. Because of their cytotoxic activity, these sesquiterpenoids have received increasing attention as a source for new anticancer drugs and pharmacophores. Natural drimanes and coloratanes, as well as their semi-synthetic derivatives, showed promising results against cancer cell lines with in vitro activities in the low micro- and nanomolar range. Despite their high potential as novel anticancer agents, the mode of action and structure–activity relationships of drimanes and coloratanes have not been completely enlightened nor systematically reviewed. Our review aims to give an overview of known structures and derivatizations of this class of sesquiterpenoids, as well as their activity against cancer cells and potential modes-of-action. The cytotoxic activities of about 40 natural and 25 semi-synthetic drimanes and coloratanes are discussed. In addition to that, we give a summary about the clinical significance of drimane and coloratane sesquiterpenoids.

No / A series of analogues of cryptolepine (1) have been synthesized and evaluated for their in vitro antiplasmodial and cytotoxic properties. The IC50 values of several compounds (11a, 11k¿m, 11o, 13) against Plasmodium falciparum (strain K1) were <0.1 ¿M, 5¿10-fold lower than that of 1 but their cytotoxicities were only 2¿4 times greater than that of 1. Compounds with a halogen in the quinoline ring and a halogen or a nitro group in the indole ring have enhanced antiplasmodial activity. In mice infected with P. berghei, the 7-bromo-2-chloro (11k) and 2-bromo-7-nitro (13) derivatives of 1 suppressed parasitemia by >90% at doses of 25 mg kg-1 day-1 with no apparent toxicity to the mice. 2,7-Dibromocryptolepine (15) was evaluated at several dose levels, and a dose-dependent suppression of parasitemia was seen (ED90 = 21.6 mg kg-1 day-1). The antimalarial mode of action of 1 appears to be similar to that of chloroquine and involves the inhibition of hemozoin formation. A number of analogues were assessed for their effects on the inhibition of ß-hematin (hemozoin) formation, and the results were compared with their antiplasmodial activities having taken account of their predicted accumulation into the acidic parasite food vacuole. No correlation was seen (r2 = 0.0781) suggesting that the potent antimalarial activity of compounds such as 15 involves other mechanisms in addition to the inhibition of hemozoin formation.

No description available.

No description available.

Arsenic is a human carcinogen associated with urinary bladder transitional cell carcinoma and other cancers. Arsenic is also a strong comutagen and cocarcinogen. One possible mode of action for arsenic carcinogenesis/cocarcinogenesis is inhibition of DNA damage repair. In laboratory animals, urinary bladder transitional cell carcinoma has only been observed in dimethylarsinic acid [DMA(V)]-exposed F344 rats. The goal of the present studies was to investigate inhibition of DNA repair as a mode of action for arsenic carcinogenesis/ cocarcinogenesis in the urinary bladder. Methods were first developed to harvest only transitional cells, the target cell type of arsenic carcinogenesis, suitable for RNA extraction or for DNA damage detection by Comet assay. Morphological studies established that DMA(V) in drinking water at 40 ppm was cytotoxic to the urothelium of Sprague-Dawley and F344 rats, and mitochondria were targeted by DAM(V). To investigate whether DMA(V) decreases the expression of DNA repair genes, mRNA levels of DNA repair genes in transitional cells were next measured in F344 rats exposed to up to 100 ppm DMA(V) in drinking water for 4 weeks. The mRNA levels of Ataxia Telangectasia mutant (ATM), X-ray repair cross-complementing group 1 (XRCC1), excision repair cross-complementing group 3/Xeroderma Pigmentosum B (ERCC3/XPB), and DNA polymerase beta genes were not altered, as measured by real time RT PCR. These results suggested either that DMA(V) affects DNA repair without affecting the baseline expression of DNA repair genes or that DMA(V) does not affect DNA repair in the bladder. Arsenic effects on DNA repair were further investigated in F344 rats given 100 ppm DMA(V) or arsenate in drinking water for 1 week. DNA damage levels in transitional cells and micronuclei frequency (MN) in bone marrow were measured. Dimethylarsinic acid did not affect in vivo cyclophosphamide-induced DNA damage, and neither DMA(V) nor arsenate inhibited in vitro repair of hydrogen peroxide- or formaldehyde-induced DNA damage, as measured by Comet assay. Neither DMA(V) nor arsenate increased MN or elevated in vivo cyclophosphamide-increased MN. These results suggest inhibition of DNA repair by arsenic, in the transitional epithelium, may not be a major mechanism responsible for carcinogensis/cocarcinogenesis in the bladder. / Ph. D.

Les perturbateurs endocriniens (PE) font l’objet d’une controverse scientifique à l’heure actuelle car, pour certains scientifiques, ces molécules n’auraient pas de seuil de toxicité et agiraient à de très faibles concentrations. Cette particularité proviendrait du fait que les PE, quelles que soient leurs origines, agissent sur les hormones et leurs récepteurs. A ce titre, ils seraient assujettis aux mêmes règles que les hormones endogènes. Leurs effets à de très faibles doses s’expliqueraient par la sensibilité des récepteurs hormonaux aux faibles fluctuations de concentrations. L’absence de seuil d’effet pour les PE s’expliquerait par différents mécanismes, dont des différences d’affinité suivant la concentration entre substrat et récepteur, qui engendreraient des effets doses non monotones. Un certain nombre de revues remettent en cause ces hypothèses et les observations qui les accompagnent. Elles indiquent notamment le fait que ces données manquent de répétitions, qu’elles sont principalement issues d’études in vitro ou sur des modèles d’organismes en développement et non-nécessairement misent en perspective avec des effets toxiques pour l’organisme. Ainsi ces données ne seraient pas suffisantes pour statuer dans ce débat.Dans ce contexte, afin d’apporter de nouvelles données expérimentales au débat, nous avons cherché à caractériser les effets aux faibles doses d’un PE de référence, le flutamide (FLU), sur l’appareil reproducteur du rat mâle adulte. Pour ce faire, nous avons conduit trois études successives au cours desquelles nous avons exposé des rats Wistar de 7 semaines à une large gamme de doses de FLU. La dose la plus forte est connue pour induire des lésions testiculaires et une atrophie de la prostate. Les autres doses correspondent à la NOAEL définie dans une étude publiée précédemment, et à cette valeur divisée par 10, 100 et 1000, ce qui correspond à des faibles doses selon la définition de l’OMS. Pour chaque étude, chaque groupe était composé de 16 animaux gavés quotidiennement pendant 28 jours. Ces études ont permis d'explorer différents paramètres. Dans le cadre de la caractérisation du mode d'action du flutamide sur le testicule (Exposition --> blocage des récepteurs aux androgènes de l'hypophyse --> augmentation de la LH circulante --> prolifération des cellules de Leydig --> Hyperplasie), nous avons montré que, pour chaque évènement clé, l’effet dose est monotone et qu’il est possible de définir un seuil. Par ailleurs, nous proposons le Pdgfd, un facteur de croissance, comme médiateur de l’effet mitogène de la LH sur les cellules de Leydig.Pour la prostate, le mode d'action du flutamide n’étant pas complètement élucidé à ces niveaux de doses, le travail a été plus exploratoire. Afin d'expliquer, aux niveaux moléculaire et cellulaire, l'origine de la baisse du poids des prostates induites par le flutamide dès 1 mg/kg/j, nous avons exploré trois hypothèses: (1) l'augmentation de l'apoptose, avec un test d'activité des caspases 3 et 9 qui montre une activité supérieure pour le groupe forte dose de 10 mg/kg/j de flutamide, (2) la diminution de la prolifération, avec un marquage ki67 qui ne montre pas de différence entre le groupe témoin et la forte dose, et enfin (3) la baisse de la fonction de sécrétion de la prostate, évaluée par un western blot dirigé contre la probasine qui montre une baisse de sa production. Dans le même temps, nous avons montré, au niveau des transcrits hépatiques (RT-qPCR), que le flutamide exerce un effet-dose monotone sur certaines enzymes impliquées dans le métabolisme des xénobiotiques. En conclusion, dans ce modèle, pour l'ensemble des paramètres explorés, sur le testicule, l’hypophyse, la prostate, le sang et le foie, nous n'avons observé que des effets à seuil et aucun effet non monotone. / The dose-response characterization of Endocrine disrupting chemicals (EDCs) toxicity is an on-going debate which is controversial when exploring the nature of the dose-response curve and the effect at the low concentration. A part of the scientific community, suggests these molecules have no toxicity threshold and act at very low concentrations. This assumption stems from the fact that EDCs, whatever their origins, act on hormones and their receptors. As such, they would be subject to the same rules as the endogenous hormones. Effects at very low doses could be explained by the sensitivity of the hormone receptor to small fluctuations in concentrations. The lack of threshold for EDCs may be explained by different mechanisms, including affinity differences according to concentrations between substrate and receptor, which would generate non-monotonic dose-responses. A number of reviews challenge this assumption and the linked observations. They argue that the data are missing replication; they are mainly from in vitro studies or conducted in developing models and not-necessarily associated with toxic effects on the organism. Thus, these data are not sufficient to rule in this debate.In this context, in order to provide new experimental data to this debate, we worked to characterize the effects at low doses of a reference EDCs, flutamide (FLU) on the reproductive system of adult male rats. In that aim, we conducted three successive studies exposing Wistar rats of 7 weeks at a wide range of Flu doses. The highest dose is known to induce testicular damage and atrophy of the prostate gland. Other doses correspond to the defined NOAEL in a study published earlier, and to this value divided by 10, 100 and 1000, corresponding to low doses according to the WHO definition. For each study, each group consisted of 16 animals exposed daily by gavage for 28 days. During characterization of flutamide mode of action on the testis (Exposition -> androgen receptors blocking in the pituitary gland -> increase in circulating LH -> Leydig cells proliferation -> Leydig cells Hyperplasia), we have shown that for every key event, dose-response curve is monotonous and we were able to define a threshold. Furthermore, we suggest that the Pdgfd, a growth factor, mediates the mitogenic effect of LH on Leydig cells.In the prostate gland, the mode of action of flutamide is not completely elucidated at these dose levels, so the work was more exploratory. To explain, at the molecular and cellular levels, the origin of prostate atrophy induced by flutamide from 1 mg/kg/day, we explored three hypotheses: (1) the increase in apoptosis, with a caspases 3 and 9 activity test, which shows a higher activity for the high dosage group of 10 mg/kg/day of flutamide, (2) the decreasing of epithelium proliferation, with Ki67 marker which shows no difference between the control group and the high dose, and finally (3) the decrease in prostate secretory function, assessed by western blot against the Probasin which shows a drop in production.At the same time, we have shown in liver that flutamide has an effect on enzymes involved in the metabolism of xenobiotics with monotonic dose-response.In conclusion, in this model, for all investigated parameters on the testis, pituitary, prostate, blood and liver, we only observed threshold effects and no non-monotonic dose-responses.