Análise morfológica in vitro da ação de antifúngicos em cepas de Fonsecaea pedrosoi

A cromoblatomicose (CBM) é uma doença causada por implantação transcutânea de várias espécies de fungos melanizados. O estado do Pará é a principal área endêmica do país, sendo Fonsecaea pedrosoi o principal agente etiológico. O tratamento desta doença não é padronizado e diversas formas de intervenção são relatadas na literatura. Por outro lado, os testes de susceptibilidade in vitro aos fármacos antifúngicos podem ajudar na escolha do esquema terapêutico e na identificação de cepas resistentes. Este trabalho tem como objetivo avaliar a susceptibilidade in vitro ao itraconazol (ITZ), ao cetoconazol, (CTZ), ao fluconazol (FCZ) e a terbinafina (TBF) em 20 isolados clínicos de Fonsecaea pedrosoi, bem como as possíveis alterações morfológicas induzidas por ITZ ou TBF na Concentração Inibitória Mínima (CIM) e em altas concentrações. Os testes de susceptibilidade foram conduzidos de acordo com as recomendações do Clinical and Laboratory Standards Institute (CLSI, documento M38-A2). As concentrações finais de ITZ, TBF e CTZ em cada teste variaram de 16 a 0.03 μg/mL e de 64 a 0.125 μg/mL para o FCZ. A CIM para cada fármaco utilizado foi obtida após cinco dias de incubação a 30°C, sendo definida como a mínima concentração do fármaco capaz de reduzir em 100% o crescimento visual do fungo quando comparado com o grupo controle. O ITZ demonstrou ser o fármaco mais efetivo in vitro contra conídios de F. pedrosoi (CIM ₉₀= 1μg/mL). A TBF apresentou baixa atividade in vitro, com 70% das cepas apresentando CIM ≥ 0.5 μg/mL. Quando se analisa morfologicamente os conídios tratados com a CIM para ITZ observa-se um aumento no diâmetro celular, a presença de conídios em processo de divisão e formação de pequenas cadeias. Na maior concentração do teste de susceptibilidade (16 μg/mL) observou-se a irregularidade no contorno celular, o desprendimento de material pigmentado da parede celular e a vacuolização. Em 32 μg/mL e 64 μg/mL notou-se a ruptura da parede celular e conídos amorfos. Não foram observadas - em nenhuma das concentrações analisadas - alterações morfológicas significativas induzidas pela TBF. Além disso, a 5-Fluorocitosina (5-FC) e o FCZ não impediram o crescimento dos conídios, mesmo em altas concentrações. No entanto, alterações ultraestruturais foram notadas após tratamento com 5-FC 64 μg/mL. Portanto, sugere-se um comportamento morfológico diferente de conídios frente ao ITZ ou TBF durante os testes de susceptibilidade in vitro. Em síntese, dentre os fármacos estudados, ITZ apresentou a melhor atividade antifúngica in vitro, enquanto a 5-FC somente provocou alterações estruturais em hifas e conídios na mais alta concentração utilizada no estudo.

Phytotoxicity of ethanolic extract of turnip leaves (raphanus sativus l.)

The study of allelopathic activity has been aim of research that evaluates mainly species used in green fertilization. Raphanus sativus L. stands out among these species, because it shows high capacity for nutrient recycling, specially nitrogen and phosphorus, what makes it an advantageous cover plant in crop rotation systems. Considering the exposed, the present study had as objective the evaluation of the allelopathic and phytotoxic potentials of different concentrations of the R. sativus leaves ethanolic extract by mean of seeds germination analyses and development of lettuce seedlings, evaluating the phytotoxicity by determination of the mitotic index of lettuce root cells, realizing the phytochemical profile and investigating the antioxidant activity. It was possible to verify that the R. sativus extract interferes in the germination index, decreasing the germinability (5 mg. mL(-1) = 9.84%; 10 mg. mL(-1) = 11.91% and 20 mg. mL(-1)= 57.51%). In the lettuce seedlings growth, the extract of this species affected the roots and hypocotyls growth. It was possible to observe phenols and total flavonoids in the extract for the concentration of 1000 mu g. mL(-1)(161mg and 83.57 mg, respectively). It was also observed, higher antioxidant activity for the concentration of 1000 mu g. mL(-1) (89.76%). In the phytotoxicity assay was observed a dose dependent effect in the mitotic index and in the cellular events during cellular division. In this study it was possible to conclude that this species has allelochemical compounds which are able to interfere directly on the stabilization and development of other species.

Stochastic models and dynamic measures for the characterization of bistable circuits

During the last few years, a great deal of interest has risen concerning the applications of stochastic methods to several biochemical and biological phenomena. Phenomena like gene expression, cellular memory, bet-hedging strategy in bacterial growth and many others, cannot be described by continuous stochastic models due to their intrinsic discreteness and randomness. In this thesis I have used the Chemical Master Equation (CME) technique to modelize some feedback cycles and analyzing their properties, including experimental data. In the first part of this work, the effect of stochastic stability is discussed on a toy model of the genetic switch that triggers the cellular division, which malfunctioning is known to be one of the hallmarks of cancer. The second system I have worked on is the so-called futile cycle, a closed cycle of two enzymatic reactions that adds and removes a chemical compound, called phosphate group, to a specific substrate. I have thus investigated how adding noise to the enzyme (that is usually in the order of few hundred molecules) modifies the probability of observing a specific number of phosphorylated substrate molecules, and confirmed theoretical predictions with numerical simulations. In the third part the results of the study of a chain of multiple phosphorylation-dephosphorylation cycles will be presented. We will discuss an approximation method for the exact solution in the bidimensional case and the relationship that this method has with the thermodynamic properties of the system, which is an open system far from equilibrium.In the last section the agreement between the theoretical prediction of the total protein quantity in a mouse cells population and the observed quantity will be shown, measured via fluorescence microscopy.

Sudden Increase and Regrowth of Fecal Coliforms and Escherichia Coli in Wastewater Biosolids After High Solids Centrifugation

Treatment plants that operate either thermophilic or mesophilic anaerobic digesters with centrifugal dewatering processes have consistently observed densities of fecal coliform and Escherichia coli, both indicator bacteria, that decrease during digestion but then increase after dewatering and storage. The increases have been characterized as two separate phenomena to explain this observation: 1) “Sudden Increase,” or SI, which is defined as the increase that occurs immediately after dewatering and 2) “regrowth,” which is defined as an increase during storage of cake samples over a period of hours or days. The SI observation appears to be more prevalent with biosolids that are generated with thermophilic processes and dewatered by centrifugation. Both thermophilic and mesophilic digesters with centrifuge dewatering processes have observed the regrowth phenomena. This research hypothesizes that the SI phenomenon is due to the presence of viable nonculturable (VNC) bacteria that are reactivated during dewatering. In other words, the bacteria were always present but were not enumerated by standard culturing methods (SCM). Analysis of the E. coli density in thermally treated solids by SCMs and quantitative real-time polymerase chain reaction (qPCR) indicated that E. coli densities are often underestimated by SCM. When analyzed with qPCR, the E. coli density after digestion can be 4-5 orders of magnitude greater than the non-detect levels identified by SCMs, which supports the non-culturable hypothesis. The VNC state describes a condition where bacteria are alive but unable to sustain the metabolic process needed for cellular division. Supplements added to culturing media were investigated to determine if the resuscitation of VNC bacteria could be enhanced. The autoinducer molecules Nhexanoyl- L-Homoserine lactone (C6-HSL), 3-oxo-N-octanoyl-L-Homoserine lactone (3-oxo- C8-HSL), and norepinephrine were unable to induce the resuscitation of VNC E. coli. Additional sampling was performed to determine if autoinducer molecules, peroxides, or other as of yet unknown inhibitory agents and toxins could be removed from biosolids during SCM. Culture media supplemented with the peroxide degrading compounds catalase, α-ketoglutaric acid, and sodium pyruvate was unable to resuscitate non-culturable E. coli. The additions of bentonite and exponential growth phase E. coli cell-free supernatant to culturing media were also unable to increase the culturability of E. coli. To remove inhibitory agents and toxins, a cell washing technique was employed prior to performing SCM; however, this cell washing technique may have increased cellular stresses that inhibited resuscitation since cell densities decreased. A novel laboratory-scale dewatering process was also investigated to determine if the SI and regrowth phenomena observed in full-scale centrifugal dewatering could be mimicked in the laboratory using a lab shearing device. Fecal coliform and E. coli densities in laboratory prepared cake samples were observed to be an order of magnitude higher than full-scale dewatered cakes. Additionally, the laboratory-scale dewatering process was able to resuscitate fecal coliforms and E. coli in stored sludge such that the density increased by 4-5 orders of magnitude from nondetect values. Lastly, the addition of aluminum sulfate during centrifuge dewatering at a full-scale utility produced an increased regrowth of fecal coliforms and E. coli that was sustained for 5 days.

CO2-induced fertilization impairment in Strongylocentrotus droebachiensis collected in the Arctic

Fertilization depends on distribution and aggregation patterns of sea urchins which influence gamete contact time and may potentially enhance their vulnerability to ocean acidification. In this study, we conducted fertilization experiments to assess the effects of selected pH scenarios on fertilization success of Strongylocentrotus droebachiensis, from Spitsbergen, Arctic. Acidification was achieved by aerating seawater with different CO2 partial pressures to represent pre-industrial and present conditions (measured ~180-425 µatm) and future acidification scenarios (~550-800, ~1,300, ~2,000 µatm). Fertilization success was defined as the proportion of successful/unsuccessful fertilizations per treatment; eggs were classified according to features of their fertilization envelope (FE), hyaline layer (HL) and achievement of cellular division. The diagnostic findings of specific pathological aberrations were described in detail. We additionally measured intracellular pH changes in unfertilized eggs exposed for 1 h to selected acidification treatments using BCECF/AM. We conclude that (a) acidified conditions increase the proportion of eggs that failed fertilization, (b) acidification may increase the risk of polyspermy due to failures in the FE formation supported by the occasional observation of multiple sperms in the perivitelline space and (c) irregular formation of the embryo may arise due to impaired formation of the HL. The decrease in fertilization success could be also related to the observed changes in intracellular pH at pCO2 ~ 1,000 µatm or higher.

Research lines in Hyperthermia at the Bioinstrumentation Laboratory of the Centre for Biomedical Technology

The Bioinstrumentation Laboratory belongs to the Centre for Biomedical Technology (CTB) of the Technical University of Madrid and its main objective is to provide the scientific community with devices and techniques for the characterization of micro and nanostructures and consequently finding their best biomedical applications. Hyperthermia (greek word for “overheating”) is defined as the phenomenon that occurs when a body is exposed to an energy generating source that can produce a rise in temperature (42-45ºC) for a given time [1]. Specifically, the aim of the hyperthermia methods used in The Bioinstrumentation Laboratory is the development of thermal therapies, some of these using different kinds of nanoparticles, to kill cancer cells and reduce the damage on healthy tissues. The optical hyperthermia is based on noble metal nanoparticles and laser irradiation. This kind of nanoparticles has an immense potential associated to the development of therapies for cancer on account of their Surface Plasmon Resonance (SPR) enhanced light scattering and absorption. In a short period of time, the absorbed light is converted into localized heat, so we can take advantage of these characteristics to heat up tumor cells in order to obtain the cellular death [2]. In this case, the laboratory has an optical hyperthermia device based on a continuous wave laser used to kill glioblastoma cell lines (1321N1) in the presence of gold nanorods (Figure 1a). The wavelength of the laser light is 808 nm because the penetration of the light in the tissue is deeper in the Near Infrared Region. The first optical hyperthermia results show that the laser irradiation produces cellular death in the experimental samples of glioblastoma cell lines using gold nanorods but is not able to decrease the cellular viability of cancer cells in samples without the suitable nanorods (Figure 1b) [3]. The generation of magnetic hyperthermia is performed through changes of the magnetic induction in magnetic nanoparticles (MNPs) that are embedded in viscous medium. The Figure 2 shows a schematic design of the AC induction hyperthermia device in magnetic fluids. The equipment has been manufactured at The Bioinstrumentation Laboratory. The first block implies two steps: the signal selection with frequency manipulation option from 9 KHz to 2MHz, and a linear output up to 1500W. The second block is where magnetic field is generated ( 5mm, 10 turns). Finally, the third block is a software control where the user can establish initial parameters, and also shows the temperature response of MNPs due to the magnetic field applied [4-8]. The Bioinstrumentation Laboratory in collaboration with the Mexican company MRI-DT have recently implemented a new research line on Nuclear Magnetic Resonance Hyperthermia, which is sustained on the patent US 7,423,429B2 owned by this company. This investigation is based on the use of clinical MRI equipment not only for diagnosis but for therapy [9]. This idea consists of two main facts: Magnetic Resonance Imaging can cause focal heating [10], and the differentiation in resonant frequency between healthy and cancer cells [11]. To produce only heating in cancer cells when the whole body is irradiated, it is necessary to determine the specific resonant frequency of the target, using the information contained in the spectra of the area of interest. Then, special RF pulse sequence is applied to produce fast excitation and relaxation mechanism that generates temperature increase of the tumor, causing cellular death or metabolism malfunction that stops cellular division

The adenomatous polyposis coli-binding protein EB1 is associated with cytoplasmic and spindle microtubules

The evolutionarily conserved protein EB1 originally was identified by its physical association with the carboxyl-terminal portion of the adenomatous polyposis coli (APC) tumor suppressor protein, an APC domain commonly mutated in familial and sporadic forms of colorectal neoplasia. The subcellular localization of EB1 in epithelial cells was studied by using immunofluorescence and biochemical techniques. EB1 colocalized both to cytoplasmic microtubules in interphase cells and to spindle microtubules during mitosis, with pronounced centrosome staining. The cytoskeletal array detected by anti-EB1 antibody was abolished by incubation of the cells with nocodazole, an agent that disrupts microtubules; upon drug removal, EB1 localized to the microtubule-organizing center. Immunofluorescence analysis of SW480, a colon cancer cell line that expresses only carboxyl-terminal-deleted APC unable to interact with EB1, demonstrated that EB1 remained localized to the microtubule cytoskeleton, suggesting that this pattern of subcellular distribution is not mediated by its interaction with APC. In vitro cosedimentation with taxol-stabilized microtubules demonstrated that a significant fraction of EB1 associated with microtubules. Recent studies of the yeast EB1 homologues Mal3 and Bim1p have demonstrated that both proteins localize to microtubules and are important in vivo for microtubule function. Our results demonstrate that EB1 is a novel component of the microtubule cytoskeleton in mammalian cells. Associating with the mitotic apparatus, EB1 may play a physiologic role connecting APC to cellular division, coordinating the control of normal growth and differentiation processes in the colonic epithelium.

Análise proteômica de Chromobacterium violaceum: acúmulo estacionário e diferencial após exposição à luz UVC

Chromobacterium violaceum is a free-living bacillus, Gram-negative commonly found in water and sand of tropical and subtropical regions. One of its main characteristic it's the ability to produce the purple pigment named violacein, that shows countless biological activities. In 2003, the genome of this organism was totally sequenced and revealed important informations about the physiology of this bacteria. However, few post-genomics studies had been accomplished. This work evaluated the protein profile of C. violaceum cultivated in LB medium at 28ºC that allowed the identification and characterization of proteins related to a possible secretion system that wasn't identified and characterized yet in C. violaceum, to the quorum sensing system, to regulatory process of transcription and translation, stress adaptation and biotechnological potential. Moreover, the response of the bacteria to UVC radiation was evaluated. The comparison of the protein profile, analyzed through 2-D electrophoresis, of the control group versus the treatment group allowed the identification of 52 proteins that arose after stress induction. The obtained results enable the elaboration of a stress response pathway in C. violaceum generated by the UVC light. This pathway, that seems to be a general stress response, involves the expression of proteins related to cellular division, purine and pirimidine metabolism, heat chock or chaperones, energy supply, regulation of biofilm formation, transport, regulation of lytic cycle of bacteriophages, besides proteins that show undefined function. Despite the response present similarities with the classic SOS response of E. coli, we still cannot assert that C. violaceum shows a SOS-like response, mainly due to the absence of characterization of a LexA-like protein in this organism

Caractérisation d’un effecteur de phosphoinositides chez le parasite de la malaria "Plasmodium falciparum"

La malaria est une maladie infectieuse causant plus de 500 000 morts chaque année. La maladie est causée par un protozoaire de la famille Plasmodium. L’apparition de souches résistantes aux traitements actuels et l’absence de vaccin efficace rendent la découverte de nouvelles cibles thérapeutiques urgente. Le parasite possède un complexe apical, un groupement de vacuoles sécrétoires spécialisées contenant les protéines responsables de l’invasion du globule rouge. Nous nous intéressons aux mécanismes gouvernant le transport intracellulaire de ces protéines et à la biogenèse du complexe apical lors de la formation des nouveaux parasites. Plus particulièrement, nous nous intéressons au rôle des phosphoinositides dans le recrutement des protéines à la membrane de l’appareil de Golgi. Par analyse bio-informatique du génome de P. falciparum, nous avons identifié plusieurs protéines effectrices liant potentiellement les phosphoinositides. Les travaux présentés dans ce mémoire concernent Mal13P1.188, une protéine possédant un domaine Pleckstrin homology. Nous proposons que Mal13P1.188 ait un rôle dans la génération du complexe apical en recrutant les protéines le constituant à la membrane du Golgi par la liaison avec les phosphoinositides. Afin de vérifier nos hypothèses, nous avons généré une lignée de parasite dont le gène de Mal13P1.188 est fusionné avec une GFP et une hémagglutinine. À l’aide de cette lignée de parasite, nous avons pu identifier Mal13P1.188 à proximité de l’appareil de Golgi lorsque les parasites étaient sous la forme schizont du cycle érythrocytaire. D’autres expériences ont permis de confirmer que le domaine Pleckstrin homology de Mal13P1.188 était capable de reconnaître les différentes formes de phosphoinositides. Finalement, d’autres travaux devront être faits sur Mal13P1.188 afin de déterminer si elle est essentielle à la survie du parasite.

Spatiotemporal mechanisms for actomyosin ring assembly and contraction in budding yeast cell division

Dissertation presented to obtain the Ph.D degree in Molecular Medicine

Humoral and cellular immune responses against Type I cysteine proteinase of Leishmania infantum are higher in asymptomatic than symptomatic dogs selected from a naturally infected population.

Canids are natural reservoirs of Leishmania infantum and have been promoted as experimental hosts to decipher the pathogenesis of human visceral leishmaniasis (VL). In this study, the presence of IgG antibodies as well as the presence of mononuclear leukocytes reactive to different cysteine proteinases (CPs) were examined in 13 L. infantum-infected dogs (six with symptoms, seven asymptomatic). Cysteine proteinases which belong to papain-like enzymes known as clan CA are the most studied CPs of parasite protozoa. These molecules are expressed by the intracellular stages of the parasite and could be immunogenic. We studied Type II CP (CPA) and Type I CP (CPB) with its long C-terminal extension (CTE) which could be highly immunogenic. We showed that the level of antibodies reactive to rCPA is low in both symptomatic and asymptomatic dogs. In contrast, when CPB and CTE were used as antigens, the level of total IgG (with IgG2 superior to IgG1) reached higher values in asymptomatic dogs than in dogs with VL. While the peripheral blood mononuclear cell (PBMC) reactivity was significant when cultured in the presence of freezed/thawed (F/T) lysate, it remained low in presence of CP although always higher for PBMC recovered from asymptomatic dogs. We showed the importance of CPB and CTE in particular as a target of immune response and their potential use for serodiagnosis in asymptomatic dogs.

E2F1 regulates cellular growth by mTOR signaling

During cell proliferation, growth must occur to maintain homeostatic cell size. Here we show that E2F1 is capable of inducing growth by regulating mTORC1 activity. The activation of cell growth and mTORC1 by E2F1 is dependent on both E2F1's ability to bind DNA and to regulate gene transcription, demonstrating that a gene induction expression program is required in this process. Unlike E2F1, E2F3 is unable to activate mTORC1, suggesting that growth activity could be restricted to individual E2F members. The effect of E2F1 on the activation of mTORC1 does not depend on Akt. Furthermore, over-expression of TSC2 does not interfere with the effect of E2F1, indicating that the E2F1-induced signal pathway can compensate for the inhibitory effect of TSC2 on Rheb. Immunolocalization studies demonstrate that E2F1 induces the translocation of mTORC1 to the late endosome vesicles, in a mechanism dependent of leucine. E2F1 and leucine, or insulin, together affect the activation of S6K stronger than alone suggesting that they are complementary in activating the signal pathway. From these studies, E2F1 emerges as a key protein that integrates cell division and growth, both of which are essential for cell proliferation.

CELLULAR AND MOLECULAR STUDY OF SUBCORTICAL BAND HETEROTOPIA IN THE MOUSE MUTANT HECO

The HeCo mouse model is characterized by a subcortical heterotopia formed by misplaced neurons normally migrating into the superficial cortical layers. The mutant mouse has a tendency to epileptic seizures. In my thesis project we discovered the mutated Eml1 gene, a member of the echinoderm microtubule-associated protein (EMAP) family, in HeCo as well as in a family of three children showing complex malformation of cortical development. This discovery formed an important step in exploring the pathogenic mechanisms underlying the HeCo phenotype. In vitro results showed that during cell division the EML1 protein is associated with the midbody and a mutated version of Eml1 highlighted an important role of the protein in the astral MT array during cell cycle. In vivo, we found that already at an early age of cortical development (E13), ectopic progenitors such as RGs (PAX6) and IPCs (TBR2) accumulate in the IZ along the entire neocortex. We demonstrated that in the VZ of the HeCo mouse, spindle orientation and cell cycle exit are perturbed. In later stages (E17), RG fibers are strongly disorganized with deep layer (TBR1) and upper layer (CUX1) neurons trapped within an ectopic mass. At P3, columns of upper layer neurons were present between the heterotopia and the developing cortex; these columns were also present at P7 but at lesser extent. Time lapse video recording (E15.5) revealed that the parameters characterizing the migration of individual neurons are not disturbed in HeCo; however, this analysis showed that the density of migrating neuron was smaller in HeCo. In conclusion, truncated EML1 is likely to play a prominent role during cell cycle but also acts on the cytoskeletal architecture altering the shape of RG fibers thus influencing the pattern of neuronal migration. The signal transduction between external cues and intracellular effector pathways through MTs may be secondary but sustains the heterotopia development and further studies are needed to clarify the impact of EML1 in progenitors versus post-mitotic cells.

Activation/division of lymphocytes results in increased levels of cytoplasmic activation/proliferation-associated protein-1: prototype of a new family of proteins.

We purified from activated T lymphocytes a novel, highly conserved, 116-kDa, intracellular protein that occurred at high levels in the large, dividing cells of the thymus, was up-regulated when resting T or B lymphocytes or hemopoietic progenitors were activated, and was down-regulated when a monocytic leukemia, M1, was induced to differentiate. Expression of the protein was highest in the thymus and spleen and lowest in tissues with a low proportion of dividing cells such as kidney or muscle, although expression was high in the brain. The protein was localized to the cytosol and was phosphorylated, which is consistent with a previous report that the Xenopus laevis ortholog was phosphorylated by a mitotically activated kinase (1 ). The cDNA was previously mischaracterized as encoding p137, a 137-kDa GPI-linked membrane protein (2 ). We propose that the authentic protein encoded by this cDNA be called cytoplasmic activation/proliferation-associated protein-1 (caprin-1), and show that it is the prototype of a novel family of proteins characterized by two novel protein domains, termed homology regions-1 and -2 (HR-1, HR-2). Although we have found evidence for caprins only in urochordates and vertebrates, two insect proteins exhibit well-conserved HR-1 domains. The HR-1 and HR-2 domains have no known function, although the HR-1 of caprin-1 appeared necessary for formation of multimeric complexes of caprin-1. Overexpression of a fusion protein of enhanced green fluorescent protein and caprin-1 induced a specific, dose-dependent suppression of the proliferation of NIH-3T3 cells, consistent with the notion that caprin-1 plays a role in cellular activation or proliferation.

Multi-scale systems analysis of plant development : beyond cellular signaling and tissue morphodynamics

Les plantes sont essentielles pour les sociétés humaines. Notre alimentation quotidienne, les matériaux de constructions et les sources énergétiques dérivent de la biomasse végétale. En revanche, la compréhension des multiples aspects développementaux des plantes est encore peu exploitée et représente un sujet de recherche majeur pour la science. L'émergence des technologies à haut débit pour le séquençage de génome à grande échelle ou l'imagerie de haute résolution permet à présent de produire des quantités énormes d'information. L'analyse informatique est une façon d'intégrer ces données et de réduire la complexité apparente vers une échelle d'abstraction appropriée, dont la finalité est de fournir des perspectives de recherches ciblées. Ceci représente la raison première de cette thèse. En d'autres termes, nous appliquons des méthodes descriptives et prédictives combinées à des simulations numériques afin d'apporter des solutions originales à des problèmes relatifs à la morphogénèse à l'échelle de la cellule et de l'organe. Nous nous sommes fixés parmi les objectifs principaux de cette thèse d'élucider de quelle manière l'interaction croisée des phytohormones auxine et brassinosteroïdes (BRs) détermine la croissance de la cellule dans la racine du méristème apical d'Arabidopsis thaliana, l'organisme modèle de référence pour les études moléculaires en plantes. Pour reconstruire le réseau de signalement cellulaire, nous avons extrait de la littérature les informations pertinentes concernant les relations entre les protéines impliquées dans la transduction des signaux hormonaux. Le réseau a ensuite été modélisé en utilisant un formalisme logique et qualitatif pour pallier l'absence de données quantitatives. Tout d'abord, Les résultats ont permis de confirmer que l'auxine et les BRs agissent en synergie pour contrôler la croissance de la cellule, puis, d'expliquer des observations phénotypiques paradoxales et au final, de mettre à jour une interaction clef entre deux protéines dans la maintenance du méristème de la racine. Une étude ultérieure chez la plante modèle Brachypodium dystachion (Brachypo- dium) a révélé l'ajustement du réseau d'interaction croisée entre auxine et éthylène par rapport à Arabidopsis. Chez ce dernier, interférer avec la biosynthèse de l'auxine mène à la formation d'une racine courte. Néanmoins, nous avons isolé chez Brachypodium un mutant hypomorphique dans la biosynthèse de l'auxine qui affiche une racine plus longue. Nous avons alors conduit une analyse morphométrique qui a confirmé que des cellules plus anisotropique (plus fines et longues) sont à l'origine de ce phénotype racinaire. Des analyses plus approfondies ont démontré que la différence phénotypique entre Brachypodium et Arabidopsis s'explique par une inversion de la fonction régulatrice dans la relation entre le réseau de signalisation par l'éthylène et la biosynthèse de l'auxine. L'analyse morphométrique utilisée dans l'étude précédente exploite le pipeline de traitement d'image de notre méthode d'histologie quantitative. Pendant la croissance secondaire, la symétrie bilatérale de l'hypocotyle est remplacée par une symétrie radiale et une organisation concentrique des tissus constitutifs. Ces tissus sont initialement composés d'une douzaine de cellules mais peuvent aisément atteindre des dizaines de milliers dans les derniers stades du développement. Cette échelle dépasse largement le seuil d'investigation par les moyens dits 'traditionnels' comme l'imagerie directe de tissus en profondeur. L'étude de ce système pendant cette phase de développement ne peut se faire qu'en réalisant des coupes fines de l'organe, ce qui empêche une compréhension des phénomènes cellulaires dynamiques sous-jacents. Nous y avons remédié en proposant une stratégie originale nommée, histologie quantitative. De fait, nous avons extrait l'information contenue dans des images de très haute résolution de sections transverses d'hypocotyles en utilisant un pipeline d'analyse et de segmentation d'image à grande échelle. Nous l'avons ensuite combiné avec un algorithme de reconnaissance automatique des cellules. Cet outil nous a permis de réaliser une description quantitative de la progression de la croissance secondaire révélant des schémas développementales non-apparents avec une inspection visuelle classique. La formation de pôle de phloèmes en structure répétée et espacée entre eux d'une longueur constante illustre les bénéfices de notre approche. Par ailleurs, l'exploitation approfondie de ces résultats a montré un changement de croissance anisotropique des cellules du cambium et du phloème qui semble en phase avec l'expansion du xylème. Combinant des outils génétiques et de la modélisation biomécanique, nous avons démontré que seule la croissance plus rapide des tissus internes peut produire une réorientation de l'axe de croissance anisotropique des tissus périphériques. Cette prédiction a été confirmée par le calcul du ratio des taux de croissance du xylème et du phloème au cours de développement secondaire ; des ratios élevés sont effectivement observés et concomitant à l'établissement progressif et tangentiel du cambium. Ces résultats suggèrent un mécanisme d'auto-organisation établi par un gradient de division méristématique qui génèrent une distribution de contraintes mécaniques. Ceci réoriente la croissance anisotropique des tissus périphériques pour supporter la croissance secondaire. - Plants are essential for human society, because our daily food, construction materials and sustainable energy are derived from plant biomass. Yet, despite this importance, the multiple developmental aspects of plants are still poorly understood and represent a major challenge for science. With the emergence of high throughput devices for genome sequencing and high-resolution imaging, data has never been so easy to collect, generating huge amounts of information. Computational analysis is one way to integrate those data and to decrease the apparent complexity towards an appropriate scale of abstraction with the aim to eventually provide new answers and direct further research perspectives. This is the motivation behind this thesis work, i.e. the application of descriptive and predictive analytics combined with computational modeling to answer problems that revolve around morphogenesis at the subcellular and organ scale. One of the goals of this thesis is to elucidate how the auxin-brassinosteroid phytohormone interaction determines the cell growth in the root apical meristem of Arabidopsis thaliana (Arabidopsis), the plant model of reference for molecular studies. The pertinent information about signaling protein relationships was obtained through the literature to reconstruct the entire hormonal crosstalk. Due to a lack of quantitative information, we employed a qualitative modeling formalism. This work permitted to confirm the synergistic effect of the hormonal crosstalk on cell elongation, to explain some of our paradoxical mutant phenotypes and to predict a novel interaction between the BREVIS RADIX (BRX) protein and the transcription factor MONOPTEROS (MP),which turned out to be critical for the maintenance of the root meristem. On the same subcellular scale, another study in the monocot model Brachypodium dystachion (Brachypodium) revealed an alternative wiring of auxin-ethylene crosstalk as compared to Arabidopsis. In the latter, increasing interference with auxin biosynthesis results in progressively shorter roots. By contrast, a hypomorphic Brachypodium mutant isolated in this study in an enzyme of the auxin biosynthesis pathway displayed a dramatically longer seminal root. Our morphometric analysis confirmed that more anisotropic cells (thinner and longer) are principally responsible for the mutant root phenotype. Further characterization pointed towards an inverted regulatory logic in the relation between ethylene signaling and auxin biosynthesis in Brachypodium as compared to Arabidopsis, which explains the phenotypic discrepancy. Finally, the morphometric analysis of hypocotyl secondary growth that we applied in this study was performed with the image-processing pipeline of our quantitative histology method. During its secondary growth, the hypocotyl reorganizes its primary bilateral symmetry to a radial symmetry of highly specialized tissues comprising several thousand cells, starting with a few dozens. However, such a scale only permits observations in thin cross-sections, severely hampering a comprehensive analysis of the morphodynamics involved. Our quantitative histology strategy overcomes this limitation. We acquired hypocotyl cross-sections from tiled high-resolution images and extracted their information content using custom high-throughput image processing and segmentation. Coupled with an automated cell type recognition algorithm, it allows precise quantitative characterization of vascular development and reveals developmental patterns that were not evident from visual inspection, for example the steady interspace distance of the phloem poles. Further analyses indicated a change in growth anisotropy of cambial and phloem cells, which appeared in phase with the expansion of xylem. Combining genetic tools and computational modeling, we showed that the reorientation of growth anisotropy axis of peripheral tissue layers only occurs when the growth rate of central tissue is higher than the peripheral one. This was confirmed by the calculation of the ratio of the growth rate xylem to phloem throughout secondary growth. High ratios are indeed observed and concomitant with the homogenization of cambium anisotropy. These results suggest a self-organization mechanism, promoted by a gradient of division in the cambium that generates a pattern of mechanical constraints. This, in turn, reorients the growth anisotropy of peripheral tissues to sustain the secondary growth.