Les glioblastomes sont des tumeurs primaires du cerveau les plus malignes. L’identification des cellules souches cancéreuses de glioblastome (CSGs) a transformé notre vision globale des glioblastomes en révélant une hiérarchie cellulaire au sein de ces tumeurs. Les CSGs sont douées de propriétés d’auto-renouvellement, de différenciation et peuvent entrer en quiescence. Elles sont considérées comme les cellules entretenant les tumeurs, responsables de leur dissémination et des rechutes après traitement. La découverte des CSGs a conduit à un changement de paradigme dans le développement des thérapies anticancéreuses, avec la nécessité de cibler dans le traitement non seulement les cellules de la masse tumorale, mais aussi les CSGs. Un criblage différentiel de la chimiothèque Prestwick réalisé au laboratoire a permis d’identifier le bisacodyl comme une molécule présentant une cytotoxicité spécifique sur les CSGs en quiescence.Cette thèse présente un travail sur la caractérisation des CSGs, la compréhension du mode d’action du bisacodyl, ainsi que l’évaluation de son potentiel thérapeutique sur un modèle 3D in intro et des modèles in vivo. / Glioblastomas are the most malignant primary brain tumors. The identification of glioblastoma stemcells (GSCs) has transformed our comprehension of those tumors by revealing a hierarchical organization. GSCs can self-renew, differentiate and enter into a quiescent state. They are considered as cells which fuel and as the main culprits of tumor relapse. The discovery of GSCs triggered a change in paradigm for cancer therapy. Indeed to gain in efficacy, therapies need to target, not only the cells forming the bulk of the tumor, but also GSCs particularly resistant and endowed with a high tumorigenic potential. Chemical screening of the Prestwick chemical library in our laboratory, unveiled bisacodyl with a specific activity on quiescent GSCs.This thesis presents work on the characterization of GSCs, study of the mode of action of bisacodyl on GSCs, as well as a preclinical evaluation of bisacodyl on a 3D model in vitro and animal models in vivo.

Les glioblastomes (GBM) sont les formes les plus agressives de tumeurs gliales avec une survie médiane des patients traités n’excédant pas 2 ans. Ce mauvais pronostic est dû, entre autres, à l’hétérogénéité de ces tumeurs avec la présence de cellules souches cancéreuses (CSCs) en prolifération ou quiescentes, particulièrement résistantes aux traitements conventionnels. Cibler ces cellules au sein du microenvironment tumoral hypoxique et acides fait donc partie des thérapies d’avenir des GBMs. Le laxatif bisacodyl a été identifié par criblage de la chimiothèque Prestwick comme un composé induisant la mort par nécrose des CSCs de glioblastome (GSCs en prolifération et en quiescence), uniquement dans des conditions de faible acidité retrouvées également au sein des tumeurs. Une activité antitumorale in vivo a également été démontrée pour ce composé. Cette thèse présente l’identification du mode d’action du bisacodyl dans les GSCs. Celui-ci implique la serine/thréonine kinase WNK1 et ses partenaires, les kinases Akt et SGK1 et des co-transporteurs Na+/HCO3- NBC. Nos résultats ont également révélé un rôle de WNK1 dans la physiopathologie des GSCs. / Glioblastoma (GBM), the most aggressive glial tumor, is currently incurable with a very short-term patient survival (< 2 years). The heterogeneity of GBM and the presence of highly resistant proliferating and quiescent cancer stem-like cells (CSCs), is largely responsible for poor prognosis in this disease. Thus, new approaches targeting glioblastoma CSCs (GSCs), within the acidic/hypoxic tumor microenvironment, are promising strategies for treating GBM. The laxative bisacodyl was identified in a high throughput screening of the Prestwick chemical library as a compound inducing necrotic cell death in proliferating and quiescent GSCs only in acidic microenvironments similar to those found in tumors. Bisacodyl was further shown to induce tumor shrinking and to increase survival in in vivo GBM models. In this thesis work, we identify bisacodyl’s mechanism of action in GSCs. This mechanism involves the serine/threonine kinase WNK1 and its signaling partners including protein kinases Akt and SGK1 and NBC Na+/HCO3- cotransporters. Our data also highlight a previously unknown role of WNK1 in GSC physiopathology.

The Tardigrada are hydrophilous, segmented, molting micrometazoans that occupy a diversity of niches in freshwater, marine, and terrestrial habitats. A sister group of the arthropods, this phylum of bilaterally symmetrical lobopods, most less than 1 mm in length, have a hemocoel, a complete digestive tract, a dorsal gonad with one or two gonoducts, and a dorsal lobed brain with a ventral nerve cord and five ganglia. About 1000 species have been described based on the morphology of sclerified structures, especially the claws and buccal-pharyngeal apparatus. Reproduction occurs through fertilized or unfertilized eggs, with individuals being either gonochoric, unisexual, or hermaphroditic, and eggs are deposited either freely or within the shed exuvium. Parthenogenesis, very frequent in limnic and terrestrial tardigrades, allows them to colonize new territories by passive dispersal of a single individual. Quiescence (cryptobiosis: anhydrobiosis, anoxybiosis, cryobiosis, and osmobiosis) and diapause (encystment and resting eggs) occur during the tardigrade life history. Ecological parameters and global distribution patterns are poorly known or understood. Methods for collection, microscopy, and culturing have been developed.

Les lymphocytes T (LT) perçoivent et intègrent en permanence des signaux solubles et cellulaires, conditionnant leur comportement et leur devenir. A l'état de repos, les propriétés des LT s'appuient sur un réseau moléculaire caractéristique, au sein duquel les facteurs de transcription FoxOs jouent un rôle majeur. En effet, ces derniers sont impliqués dans le maintien de la quiescence et de la capacité circulatoire des LT, de par le profil transcriptionnel qu'ils induisent. Nous avons identifié Fam65b comme une nouvelle cible transcriptionnelle de FOXO1. D'un point de vue fonctionnel, nous avons démontré que Fam65b régule négativement le seuil de prolifération des LT en réponse à une stimulation du récepteur à l'antigène (TCR) ou du récepteur aux chimiokines CCR7. In vivo, dans un modèle de souris transgénique pour le TCR, ces caractéristiques fonctionnelles se traduisent par une réponse secondaire plus efficace en absence de Fam65b. Physiologiquement, la moindre expression de Fam65b que nous avons observé dans les LT mémoires par comparaison aux LT naïfs corrèle avec leur plus grande réactivité. L'ensemble de ces résultats suggère que Fam65b pourrait être un marqueur fonctionnel des LT mémoires. Enfin, nous avons pu démontrer que les effets fonctionnels de Fam65b résultent d'une inhibition de l'activité de RhoA.Fam65b est donc un régulateur de la quiescence et de la migration des LT. De par son rôle de régulateur de l'activité de RhoA, Fam65b constitue un nouveau lien fonctionnel entre deux familles majeures, contrôlant la physiologie des LT : les Rho-GTPases et les FoxOs.

Le muscle squelettique adulte est capable de se régénérer à plusieurs reprises après blessure grâce à sa population de cellules souches résidentes: les cellules satellites. Cependant, les mécanismes impliquant les cellules satellite dans la recouvrement de l'homéostasie et de l'intégrité musculaire ne sont toujours pas clairs. Chez l'adulte, les cellules satellites sont quiescentes et localisées dans une niche entre la lame basale et la fibre musculaire. Après blessure, elles prolifèrent, se différencient et fusent afin de restaurer les fibres endommagées. Lorsque la niche des cellules satellite est altérée elles expriment rapidement le marqueur d'activation Myod puis prolifèrent. La lame basale des cellules souches est riche en collagène, glycoprotéines et de protéoglycan. Cependant, le mécanisme de fonction de ces protéines de la matrice extracellulaire (MEC) dans le maintien de la cellule satellite dans sa niche est toujours inconnu. De plus, l'interaction entre la MEC et des voies de signalisation cellulaire essentielles au maintien des cellules souches quiescentes sont toujours un mystère. Nous avons identifiés la voie Notch comme effecteur indispensable à la quiescence des cellules satellites. Un ChIP screening dans des cellules musculaires nous a permit d'identifier des microRNAs et collagènes spécifiques comme des gènes cibles de la voie Notch. L'utilisation d'outils génétiques permettant de moduler l'activité de la voie Notch démontrent que ces microRNAs et collagènes sont régulés transcriptionnellement par la voie Notch in vitro et in vivo. Nous proposons que le Collagène de type V et miR-708, induits par Notch, peuvent autoréguler la niche des cellules souches. / Adult skeletal muscles can regenerate after repeated trauma, yet our understanding of how adult muscle satellite (stem) cells (MuSCs) restore muscle integrity and homeostasis after regeneration is limited. In the adult mouse, MuSCs are quiescent and located between the basal lamina and the myofibre. After injury, they re-enter the cell cycle, proliferate, differentiate and fuse to restore the damaged fibre. A subpopulation of myogenic cells then self-renews and replenishes the stem cell pool for future repair. When MuSCs are removed from their niche, they rapidly express the commitment marker Myod and proliferate. The basal lamina that ensheaths MuSCs is rich in collagens, non-collagenous glycoproteins and proteoglycans. Whether these and other extracellular matrix (ECM) proteins constitute functional components of MuSCs niche remains unclear. Moreover, although signalling pathways that maintain MuSCs quiescence have been identified, how these regulate stem cell properties and niche composition remains largely unknown. Sustained, high activity of the Notch signalling pathway is critical for the maintenance of MuSCs in a quiescence state. Of interest, whole-genome ChIP for direct Notch/Rbpj transcriptional targets identified specific micro-RNAs and collagen genes in satellite cells. Using genetic tools to conditionally activate or abrogate Notch signalling, we demonstrate that the expression of these target genes is controlled by the Notch pathway in vitro and in vivo. Further, we propose that Collagen V and miR708 can contribute cell-autonomously to the generation of the MuSCs niche via a Notch signalling-regulated mechanism.

During aging there is a decline in (MuSCs) and muscle regeneration, though the underlying reason is unknown. Interestingly, mitochondrial fragmentation is a common feature in aging, however, how this impacts MuSC function and maintenance has not been investigated. To address the effect of mitochondrial fragmentation in MuSCs, we generated a knockout mouse model using the Pax7CreERT2 inducible system to target deletion of the mitochondrial fusion protein Opa1 specifically within MuSCs (Opa1-KO). Analysis of MuSC function following muscle injury revealed a defect in the regenerative potential of Opa1-KO MuSCs. Moreover, following injury there was a substantial decrease in the number of MuSC in Opa1-KO animals with a concomitant increase in the number of committing cells, illustrating that loss of Opa1 drives MuSC towards commitment at the expense of self-renewal. Furthermore, loss of Opa1 in MuSCs alters the quiescence state, priming MuSCs for activation, as indicated by a reduction in quiescence-related genes, increased EdU incorporation, and enhanced cell cycle kinetics. To address the impact of mitochondrial dysfunction on muscle stem cell capacity, we generated a model of chronic Opa1 loss. Analysis of muscle stem cell function 3 months after Opa1 ablation revealed mitochondrial dysfunction and a defect in proliferation upon activation, leading to failed muscle regeneration. These data are the first to demonstrate a novel role for mitochondrial structure in the regulation of MuSC maintenance and regenerative capacity.

Recent research identifies a number of factors associated with public support for or opposition to environmentally-contentious energy infrastructure projects. Much of that research documents the attitudes of populations surrounding projects where energy is produced, such as powerhouses, mines, or drilling operations. I use survey and interview data to argue that those factors do not adequately reflect the concerns of landowners distributed along the 303-mile path of a rural Appalachian natural gas project, which I identify as a site of energy transmission rather than production. I use social representation theory to elicit factors unrecognized in prior research. It provides a framework for the process by which resident rural landowners become aware of, interpret, evaluate, and then respond to the Mountain Valley Pipeline. Landowners express their sense of injustice when the pipeline developer, public policymakers, and permitting authorities are unaware of or indifferent to factors that are especially relevant to them as the pipeline is imposed on their rural environment. The study is based on a sequential mixed-methods approach. I conducted a secondary analysis of the Quality of Life in Rural Virginia and West Virginia Survey dataset (Bell et al. 2019), which consists of mail surveys completed by 783 residents living in 10 counties along the route of the Mountain Valley Pipeline. In 2021 and 2022 I conducted follow-up semi-structured interviews with 25 landowners in the blast zone, which is 1,115 feet on either side of this pipeline, who had completed the survey. The first aim was to test three factors that prior research suggested are associated with attitudes toward such projects. The first factor, economic self-interest, was statistically nonsignificant for these landowners. The interview data suggest that unlike sites of energy production, where jobs stimulate support, landowners saw few jobs available for local people. Any financial value from the sale of easements did not affect their support. The second factor, political ideology, was important in other studies, because conservative ideology is associated with pro-business attitudes. In contrast, even though 60% of the landowners in this study identified themselves as conservative, there was only a weak association between political ideology and support for the pipeline, due in part to the perception of inappropriate application of eminent domain law by the pipeline developer and the courts. Distance from the pipeline, the third factor, was moderately associated with attitude toward the project, with less support for pipeline construction among landowners in the blast zone. The second aim was to use social representation theory to reveal factors in addition to distance that influenced landowners' attitudes toward the project. Interviews revealed that landowners in the blast zone were as concerned with threats to cherished water supplies, for both domestic and agricultural uses, as they were with the danger of a pipeline explosion. The interviews also revealed participants' concern for the disruption of their attachment to and dependence on their properties. These factors were underrepresented in the planning and permitting for this project. The intuitive, common-sense structure for eliciting landowners' attitudes provided by social representation theory was effective at this microscale of inquiry, and may be useful for comparative studies that further distinguish between sites of energy production and sites of energy transmission. / Doctor of Philosophy / Recent research identifies a number of factors associated with public support for or opposition to environmentally-contentious energy infrastructure projects. Much of that research documents the attitudes of populations surrounding projects where energy is produced, such as powerhouses, mines, or drilling operations. I use survey and interview data to argue that those factors do not adequately reflect the concerns of landowners distributed along the 303-mile path of a rural Appalachian natural gas project, which I identify as a site of energy transmission rather than production. I use social representation theory to elicit factors unrecognized in prior research. It provides a framework for the process by which resident rural landowners become aware of, interpret, evaluate, and then respond to the Mountain Valley Pipeline. Landowners express their sense of injustice when the pipeline developer, public policymakers, and permitting authorities are unaware of or indifferent to factors that are especially relevant to them as the pipeline is imposed on their rural environment. The study is based on a sequential mixed-methods approach. I conducted a secondary analysis of the Quality of Life in Rural Virginia and West Virginia Survey dataset (Bell et al. 2019), which consists of mail surveys completed by 783 residents living in 10 counties along the route of the Mountain Valley Pipeline. In 2021 and 2022 I conducted follow-up semi-structured interviews with 25 landowners in the blast zone, which is 1,115 feet on either side of this pipeline, who had completed the survey. Survey data suggest that factors, including economic self-gain, political ideology, and proximity to the pipeline, differ from predictions reported in prior research. Interview data suggest that in this case study those differences are associated with landowner attitudes toward danger and disruption to their sense of place. These differences may be specifically applicable to rural populations exposed to sites of energy transmission rather than sites of energy production. A recognition of these differences has important implications for project developers, public policy planners, and permitting agencies.

Les cellules dendritiques sont des cellules du système immunitaire inné qui jouent un rôle important dans la reconnaissance immunitaire contre les agents pathogènes étrangers. Elles peuvent également prévenir les maladies auto-immunes à l'état basal. En raison de l'importance des cellules dendritiques dans la régulation immunitaire, il est important de comprendre comment le développement, l'état d'homéostasie et de quiescence des ces cellules sont contrôlées dans des conditions physiologiques et pathologiques. Cette étude permettra non seulement de mieux comprendre le contrôle de la régulation immunitaire, mais aussi de contribuer au développement de nouvelles approches pour traiter les maladies infectieuses et auto-immunes, ainsi que les cancers. Notre laboratoire a montré que C-CBL et CBL-B, deux membres de la famille CBL des ubiquitine ligases E3, jouent un rôle redondant dans la régulation négative du développement et de l'activation des cellules T et B. En l'absence de CBL dans les cellules T ou B, les souris développent des maladies auto-immunes sévères, indiquant que C-CBL et CBL-B jouent un rôle dans le système auto-immun. Partant de ces observations, nous proposons que CBL-B et C-CBL peuvent également jouer un rôle similaire dans le développement et la fonction des cellules dendritiques. Pour étudier cette possibilité, nous avons généré une souris knockout de Cbl spécifiques aux cellules dendritiques (dKO). Nous avons trouvé que cette mutation provoque une modification de l'homéostasie d'un sous-ensemble des cellules dendritiques (DC), y compris une augmentation marquée des CD8a+ cDCs et une réduction des pDC dans la rate. Cette modification est causée par la prolifération accrue des CD8a+ cDCs. Dans les CD8a+ cDCs mutantes, les voies de signalisation PKB et ERK sont constitutivement activées. Blocage de la signalisation de MTOR par la rapamycine atténue de manière significative l'hyperprolifération des CD8a+ cDCs in vitro et in vivo, indiquant que l'hyperactivation de MTOR est en partie responsable de l'augmentation CD8a+ cDCs. Les protéines CBL contrôlent l'ubiquitination et la dégradation du récepteur FLT3, suggérant que les protéines CBL contrôlent ainsi l'homéostasie de CD8a+ cDCs. Outre ces effets sur le développement des cellules dendritiques, nous avons trouvé que les souris Cbl dKO développent des inflammations sévères du foie et d'autres organes, caractérisées par une infiltration massive de leucocytes et une activation importante des cellules lymphocytes T périphériques. Les souris mutantes produisent des niveaux élevés de cytokines inflammatoires et de chimiokines, telles que le TNF-α, l'IL-6 et le CCL2. Les souris mutantes développent une maladie inflammatoire du foie. L'ensemble de ces observations montrent que les protéines CBL jouent un rôle essentiel dans le maintien de la quiescence immunitaire chez la souris. Puisque les souris dKO Cbl développent principalement une inflammation sévère du foie, il serait intéressant d'étudier si les voies contrôlées par les protéines CBL contribuent également au développement d'une inflammation du foie chez l'homme. / Dendritic cells (DCs) are innate immune cells that play an important role in immune recognition against foreign pathogens. They may also sense self-cues and prevent autoimmune diseases under the steady-state. Given the importance of DCs in immune regulation, it is conceivable that understanding how DCs development, homeostasis and functional quiescence are regulated under physiological and pathological conditions will not only bring insight into our knowledge how immune regulation is controlled but also some new approaches to treat infectious and autoimmune diseases and even cancers. Dr. Gu’s lab previously has shown that C-CBL and CBL-B, two members of the CBL family of E3 ubiquitin ligases, play a redundant negative regulatory role in both T cells and B cells development and activation. In the absence of CBL family of proteins in either T or B cells, mice develop severe autoimmune diseases, indicating that C-CBL and CBL-B restrain immune system against self. Based on these discoveries, we propose that C-CBL and CBL-B may also have a similar regulatory role in DC development and function. To study this possibility, we have generated DC-specific Cbl dKO mice. We have found that the Cbl dKO mutation results in an altered homeostasis of DC subsets, including a marked increase of CD8a+ cDCs and reduction of pDCs in the spleen (SP). This alteration is due to the enhanced proliferation of CD8a+ cDCs rather than the preferential lineage commitment to CD8a+ cDCs. In the mutant CD8a+ cDCs, both the PKB signaling pathway and ERK signaling pathways are constitutively activated. Blockage of MTOR signaling by Rapamycin significantly attenuates the hyperproliferation of CD8a+ cDCs both in vitro and in vivo, indicating that hyperactivation of MTOR is at least one of the reasons leading to CD8a+ cDC expansion. CBL proteins regulate ubiquitination and degradation of FLT3. Based on these results, we conclude that CBL proteins control CD8a+ cDC homeostasis through promoting FLT3 ubiquitination and degradation. In addition to the altered DC development, we have found that Cbl dKO mice develop severe liver and other organ inflammation characterized by massive leukocytes infiltration and profound peripheral T cell activation. Mutant mice produce high levels of inflammatory cytokines and chemokines including TNF-a, IL-6, CCL2, etc. Most strikingly, the mutant mice develop a similar liver inflammatory disease even in the absence of T and B cells. These findings together indicate that CBL proteins play an essential role in the maintenance of immune quiescence in mice. Since Cbl dKO mice mainly develop severe liver inflammation, it will be interesting to study whether the pathways controlled by CBL proteins also contribute to the development of liver inflammation in humans.

O objetivo do trabalho foi determinar o efeito da temperatura e período de molhamento no processo de infecção de Colletotrichum gloeosporioides e C. acutatum em goiaba e folhas de citros, respectivamente, além de evidenciar o processo de colonização de C. gloeosporioides. Para determinar o processo de infecção em diferentes combinações de temperatura e períodos de molhamento, suspensões de conídios de C. gloeosporioides foram depositadas em placas de poliestireno e incubadas sob temperaturas de 10, 15, 20, 25, 30, 35 e 40 °C, com período de molhamento de 6, 12, 24, 36 e 48 horas. Para C. acutatum, as placas foram incubadas sob temperaturas de 5, 10, 15, 20, 25, 30 e 35 °C, com períodos de molhamento de 6, 12, 24, 36 e 48 horas. In vivo, suspensões de conídios de C. gloeosporioides foram depositadas na superfície de goiabas que foram incubadas sob temperaturas de 10, 15, 20, 25 e 30 °C e períodos de molhamento de 6, 12 e 24 horas. Folhas de citros foram inoculadas com suspensões de dois isolados de C. acutatum e incubadas sob temperatura de 15, 20, 25 e 30 °C e períodos de molhamento de 12, 24 e 48 horas. Para os estudos do processo de colonização, goiabas com 110 dias após a queda das pétalas foram inoculadas e incubadas a 25 °C e períodos de molhamento de 48, 72, 96 e 120 horas. Posteriormente, frutos com 10, 35, 60 e 85 dias também foram inoculados e incubados a 25 °C por 48 horas. Para visualizar estruturas do tecido vegetal e fenóis, secções de frutos com as diferentes idades foram coradas com azul de toluidina e ACN. As temperaturas ótimas in vitro para germinação de C. gloeosporioides, apressórios formados e melanizados foram, respectivamente, 22,7, 20,6 e 23 °C. Para o isolado KLA-MGG-1 de C. acutatum foi 23,9 °C para germinação e 23,5 °C para formação de apressórios, enquanto para o isolado FSH-CLB-2 foi 21,6 °C para ambas as variáveis. Em goiaba, as temperaturas ótimas para germinação de C. gloeosporioides e formação de apressórios foram 22,4 e 23,3 °C, respectivamente. Em folhas de laranjeira, as temperaturas ótimas para os isolados KLA-MGG-1 e FSH-CLB-2 foram, respectivamente, 24,1 e 24 °C para germinação e 21,2 e 23 °C para formação de apressórios. Para folhas de limoeiro, foram 18,1 °C para germinação e 16,2 °C para formação de apressórios do isolado KLA-MGG-1. Para o isolado FSH-CLB-2, as temperaturas ótimas foram 24,4 e 23,7 °C, respectivamente. A estratégia de colonização de C. gloeosporioides foi intracelular hemibiotrófica. Em amostras com 48 h após a inoculação, foi verificado o peg de penetração. Com 72 horas, observou-se a formação da vesícula de infecção. As hifas foram observadas em amostras com 96 h após inoculação. As mesmas estruturas fúngicas alcançaram as células parenquimáticas com 120 horas após inoculação. O peg de penetração foi observado apenas em frutos com 85 e 110 dias. Estruturas do tecido vegetal e fenóis foram alterados com a idade dos frutos. / The objective of this study was to determine the effect of temperature and the wetness periods in the infection process of Colletotrichum gloeosporioides and C. acutatum in guava and citrus leaves, respectively, besides evidencing the colonization process of C. gloeosporioides. To determine the infection process at different temperature and wetness periods combinations, conidial suspensions of C. gloeosporioides were deposited on polystyrene dishes and incubated at 10, 15, 20, 25, 30, 35 and 40 °C with wetness periods of 6, 12, 24, 36 and 48 h. For C. acutatum, the dishes were incubated at 5, 10, 15, 20, 25, 30 and 35 °C, with wetness periods of 6, 12, 24, 36 and 48 h. In vivo conidial suspensions of C. gloeosporioides were placed on the surface of guavas which were incubated at 10, 15, 20, 25 and 30 °C with wetness periods of 6, 12 and 24 h. The citrus leaves were inoculated with suspensions of two isolates of C. acutatum and incubated at 15, 20, 25 and 30 °C with wetness durations of 12, 24 and 48 h. For the analysis on the colonization process, physiological mature guava fruits were inoculated and incubated at 25 °C with wetness periods of 48, 72, 96 and 120 h. Afterward, fruits with 10, 35, 60 and 85 days were also inoculated and incubated at 25 °C for 48 hours. To visualize the structures of vegetal tissues and phenols, sections of fruits at different ages were colored in toluidine blue and ACN. Optical temperature for conidial germination, appressoria formation and appressoria melanization for C. gloeosporioides were, respectively, 22.7, 20.6 and 23.0 °C. For C. acutatum isolate KLA-MGG-1, they were 23.9 °C for germination and 23.5 °C for appressoria formation and for isolate FSH-CLB-2 it was 21.6 °C for both variable. In guava, the temperatures for germination of C. gloeosporioides and appressoria formation were 22.4 and 23.3 °C, respectively. In leaves of orange trees, the optimal temperatures for the isolates KLA-MGG-1 and FSH-CLB-2 were, respectively, 24.1 and 24 °C for germination and 21.2 and 23 °C for appressoria formation. In leaves of lemon trees, they were 18.1 °C for germination and 16.2 °C for appressorial production of isolate KLA-MGG-1. For isolate FSH-CLB-2, the optimal temperatures were 24.4 and 23.7 °C, respectively. The colonization strategy of C. gloeosporioides was intracellular hemibiotrophic. The penetration peg was verified in samples 48 h after inoculation. After 72 h, it was observed formation of infection vesicle. The hyphae were observed in samples 96 h after inoculation. The same fungal structures reached the parenchymal cells 120 hours after inoculation. The penetration peg was observed only in fruits with 85 and 110 days. Structures of guava tissues and phenols were changed with the fruit aging.

O objetivo do trabalho foi determinar o efeito da temperatura e período de molhamento no processo de infecção de Colletotrichum gloeosporioides e C. acutatum em goiaba e folhas de citros, respectivamente, além de evidenciar o processo de colonização de C. gloeosporioides. Para determinar o processo de infecção em diferentes combinações de temperatura e períodos de molhamento, suspensões de conídios de C. gloeosporioides foram depositadas em placas de poliestireno e incubadas sob temperaturas de 10, 15, 20, 25, 30, 35 e 40 °C, com período de molhamento de 6, 12, 24, 36 e 48 horas. Para C. acutatum, as placas foram incubadas sob temperaturas de 5, 10, 15, 20, 25, 30 e 35 °C, com períodos de molhamento de 6, 12, 24, 36 e 48 horas. In vivo, suspensões de conídios de C. gloeosporioides foram depositadas na superfície de goiabas que foram incubadas sob temperaturas de 10, 15, 20, 25 e 30 °C e períodos de molhamento de 6, 12 e 24 horas. Folhas de citros foram inoculadas com suspensões de dois isolados de C. acutatum e incubadas sob temperatura de 15, 20, 25 e 30 °C e períodos de molhamento de 12, 24 e 48 horas. Para os estudos do processo de colonização, goiabas com 110 dias após a queda das pétalas foram inoculadas e incubadas a 25 °C e períodos de molhamento de 48, 72, 96 e 120 horas. Posteriormente, frutos com 10, 35, 60 e 85 dias também foram inoculados e incubados a 25 °C por 48 horas. Para visualizar estruturas do tecido vegetal e fenóis, secções de frutos com as diferentes idades foram coradas com azul de toluidina e ACN. As temperaturas ótimas in vitro para germinação de C. gloeosporioides, apressórios formados e melanizados foram, respectivamente, 22,7, 20,6 e 23 °C. Para o isolado KLA-MGG-1 de C. acutatum foi 23,9 °C para germinação e 23,5 °C para formação de apressórios, enquanto para o isolado FSH-CLB-2 foi 21,6 °C para ambas as variáveis. Em goiaba, as temperaturas ótimas para germinação de C. gloeosporioides e formação de apressórios foram 22,4 e 23,3 °C, respectivamente. Em folhas de laranjeira, as temperaturas ótimas para os isolados KLA-MGG-1 e FSH-CLB-2 foram, respectivamente, 24,1 e 24 °C para germinação e 21,2 e 23 °C para formação de apressórios. Para folhas de limoeiro, foram 18,1 °C para germinação e 16,2 °C para formação de apressórios do isolado KLA-MGG-1. Para o isolado FSH-CLB-2, as temperaturas ótimas foram 24,4 e 23,7 °C, respectivamente. A estratégia de colonização de C. gloeosporioides foi intracelular hemibiotrófica. Em amostras com 48 h após a inoculação, foi verificado o peg de penetração. Com 72 horas, observou-se a formação da vesícula de infecção. As hifas foram observadas em amostras com 96 h após inoculação. As mesmas estruturas fúngicas alcançaram as células parenquimáticas com 120 horas após inoculação. O peg de penetração foi observado apenas em frutos com 85 e 110 dias. Estruturas do tecido vegetal e fenóis foram alterados com a idade dos frutos. / The objective of this study was to determine the effect of temperature and the wetness periods in the infection process of Colletotrichum gloeosporioides and C. acutatum in guava and citrus leaves, respectively, besides evidencing the colonization process of C. gloeosporioides. To determine the infection process at different temperature and wetness periods combinations, conidial suspensions of C. gloeosporioides were deposited on polystyrene dishes and incubated at 10, 15, 20, 25, 30, 35 and 40 °C with wetness periods of 6, 12, 24, 36 and 48 h. For C. acutatum, the dishes were incubated at 5, 10, 15, 20, 25, 30 and 35 °C, with wetness periods of 6, 12, 24, 36 and 48 h. In vivo conidial suspensions of C. gloeosporioides were placed on the surface of guavas which were incubated at 10, 15, 20, 25 and 30 °C with wetness periods of 6, 12 and 24 h. The citrus leaves were inoculated with suspensions of two isolates of C. acutatum and incubated at 15, 20, 25 and 30 °C with wetness durations of 12, 24 and 48 h. For the analysis on the colonization process, physiological mature guava fruits were inoculated and incubated at 25 °C with wetness periods of 48, 72, 96 and 120 h. Afterward, fruits with 10, 35, 60 and 85 days were also inoculated and incubated at 25 °C for 48 hours. To visualize the structures of vegetal tissues and phenols, sections of fruits at different ages were colored in toluidine blue and ACN. Optical temperature for conidial germination, appressoria formation and appressoria melanization for C. gloeosporioides were, respectively, 22.7, 20.6 and 23.0 °C. For C. acutatum isolate KLA-MGG-1, they were 23.9 °C for germination and 23.5 °C for appressoria formation and for isolate FSH-CLB-2 it was 21.6 °C for both variable. In guava, the temperatures for germination of C. gloeosporioides and appressoria formation were 22.4 and 23.3 °C, respectively. In leaves of orange trees, the optimal temperatures for the isolates KLA-MGG-1 and FSH-CLB-2 were, respectively, 24.1 and 24 °C for germination and 21.2 and 23 °C for appressoria formation. In leaves of lemon trees, they were 18.1 °C for germination and 16.2 °C for appressorial production of isolate KLA-MGG-1. For isolate FSH-CLB-2, the optimal temperatures were 24.4 and 23.7 °C, respectively. The colonization strategy of C. gloeosporioides was intracellular hemibiotrophic. The penetration peg was verified in samples 48 h after inoculation. After 72 h, it was observed formation of infection vesicle. The hyphae were observed in samples 96 h after inoculation. The same fungal structures reached the parenchymal cells 120 hours after inoculation. The penetration peg was observed only in fruits with 85 and 110 days. Structures of guava tissues and phenols were changed with the fruit aging.