Os conhecimentos sobre os mecanismos regulatórios responsáveis pelo crescimento dos fungos filamentosos apresentam lacunas e sua compreensão é necessária para o desenvolvimento de uma terapêutica antifúngica mais adequada, assim como para incrementar a síntese de produtos de interesse comercial. Assim sendo, estudar o envolvimento do Ca2+ na resposta de um fungo modelo como A. nidulans sob fontes de carbono diferentes constitui um meio de gerar conhecimentos sobre as características de crescimento dos fungos filamentosos, de sua resposta a adaptação ambiental e dos mecanismos que controlam essa resposta. Analisou-se na linhagem A26 e na AP27, esta última com ruptura do gene da plcA, o gradiente de Ca2+ citosólico, a morfologia das hifas, a germinação e o ciclo de divisão nuclear quando as linhagens tinham calcineurina ou calmodulina inibidas e quando os canais de Ca2+ estavam bloqueados ou abertos. Os níveis de Ca2+ citosólico na linhagem A26, crescendo em presença de glicose, foram maiores que os detectados em meio suplementado com pectina. O ciclo de germinação e divisão celular no AP27, independentemente da fonte de carbono, mostrou-se mais lento se comparado com a linhagem A26, provavelmente devido ao fato de seus estoques intracelulares de Ca2+, tanto em nível vesicular quanto citosólico, serem menores. A linhagem AP27 apresentou ramificações dicotômicas nas pontas das hifas e nas hifas laterais em ambas as fontes de carbono nas quais foi cultivada, o que não se observou na linhagem A26. Quando calcineurina foi inibida por ciclosporina A, as hifas das duas linhagens, em ambas condições de cultivo, alongaram-se menos e apresentaram-se mais ramificadas, no entanto este efeito foi mais pronunciado em presença de glicose, e entre as duas linhagens pode-se dizer que foi mais intenso na linhagem AP27, demonstrando a importância dos níveis de cálcio na atividade desta enzima e conseqüentemente no desenvolvimento normal das hifas. A abertura dos canais de Ca2+, por ionóforo, produziu hiperramificação em ambas as linhagens, mas principalmente quando cresciam em pectina e ao contrário do efeito observado em presença de verapamil, que bloqueia os canais de Ca2+, não promoveram hifas laterais e nem pontas dicotômicas. No entanto o outro bloqueador dos canais de Ca2+ testado, ácido caurenóico, apresentou efeito morfológico diferente, pois as hifas tornaram-se curvas o que indica perda de polaridade. O inibidor da calmodulina (TFP) retardou a germinação, principalmente no mutante AP27, quando crescendo em presença de glicose. Lembrando que o complexo Ca2+/CaM ativa a calcineurina e que o mutante apresenta menores níveis de cálcio, esse resultado é justificável. A ruptura do gene plcA não impediu o crescimento e desenvolvimento do mutante, provavelmente porque a função desta enzima poder ser provida por outras partes do genoma, mas comprometeu os níveis intracelulares de cálcio e conseqüentemente a sua morfologia. Este estudo mostra a importância da fosfolipase C, para manutenção dos níveis intracelulares de Ca2+, no desenvolvimento normal das hifas de A. nidulans e, pela primeira vez, demonstra que esses níveis são diferentes quando o fungo cresce em presença de uma fonte de carbono, prontamente metabolizável ou não. Esses resultados conferem ao cálcio um papel modulador nessas condições de cultivo. / The knowledge about regulatory mechanisms responsible for filamentous fungi growth presents lacks and its understanding is important to develop adequate antifungal therapy either to contribute the synthesis of interestingly commercial products. By this way, study Ca2+ relationship to a fungal model as A. nidulans about different carbon sources, constitute knowledge about the filamentous fungi growing characteristics, environment adaptation and its control mechanisms. The strains A26 and AP27 was analyzed, this last one with disruption plcA gene, cytosolic Ca2+ gradient hyphal morfology, germination and nuclear division cycle when these strains had calcineurin or calmodulin inhibition and Ca2+ channel were blocked or opened. Cytosolic Ca2+ levels in A26 strain, growing in the presence of glucose was higher than supplemented media with pectin. AP27 strain, independently of carbon source, demonstrated lower germination and cell division than A26 strains, probably due to the fact that intracellular Ca2+ stocks either vesicular as cytosolic levels were lower. AP27 strain presented dichotomous branching at tip-high and lateral hyphae, at both carbon source that was grown, didnt observed at A26 linkage. When calcineurin was inhibited by cyclosporin A, hyphae from both strains, in both growth conditions, had less elongated and showed more branching, however this effect was more pronounced in presence of glucose, and between both strains was more intense at AP27 strain, indicating the importance of Ca2+ levels at this enzymatic activity and therefore the normal development of hyphae. The opening of Ca2+ channel by ionophore, produced hyperbranching in both strains, even when growth in pectin and in contrast of effect observed in the presence of verapamil, that blocks Ca2+ channels, didnt promote lateral or tip high dichotomous branching. However kaurenoic acid, an another Ca2+ channel blocker tested, presented different morphological effect, because hyphae became curved, indicating loss of polarity. Calmodulin inhibitor (TFP) delayed germination mainly at mutant AP27, when growing in the presence of glucose. Remembering that Ca2+/CaM complex activate calcineurin and the mutant exhibit lower Ca2+ levels, justifying this results. The rupture of plcA gene didnt affected growth and development of mutant, probably because the function of this enzyme can be provided by another parts of genoma, damaged the Ca2+ intracellular levels and consequently its morphology. This study shows the importance of fosfolipase C to maintaining the intracellular Ca2+ levels, at the normal hyphae development of A. nidulans and for the first time, demonstrating that this levels are different when fungi are grown in the presence of carbon source, promptly metabolizable or not. This results gives to Ca2+ as modulator at growth conditions.

The four main steps of bacterial viruses (bacteriophages) lytic infection are (i) specific recognition and genome entry into the host bacterium, (ii) replication of the viral genome, (iii) assembly of viral particles, and (iv) their release, leading in most cases to cell lysis. Although the course of individual steps of the viral infection cycle has been relatively well established, the details of how viral DNA transits from the virion to the host cytoplasm and of how the cellular environment catalyzes and possibly organizes the entire process remain poorly understood.Tailed bacteriophages are by far the most abundant viruses that infect Eubacteria. The first event in their infection is recognition of a receptor on the surface of host bacterium by the phage adsorption machinery. The barriers that the infectious particle overcomes subsequently are the cell wall and the cytoplasmic membrane of bacteria. This implies a localized degradation of the wall and the flow of its double stranded DNA (dsDNA) through a hydrophilic pore in the membrane. The lineards DNA molecule is most frequently circularized in the cytoplasm followed by its replication. In this study we used bacteriophage SPP1 that infects the Gram-positive bacterium Bacillus subtilis as a model system to dissect the different steps leading to transfer of the phage genome from the viral capsid to the host cell cytoplasm.normally to B. subtilis but do not trigger depolarization of the CM. Attachment of intact SPP1 particles is thus required for phage-induced depolarization.The beginning of B. subtilis infection by bacteriophage SPP1 was followed inspace and time. The position of SPP1 binding at the cell surface was imaged by fluorescence microscopy using virus particles labeled with "quantum dots". We found that SPP1 reversible adsorption occurs preferentially at the cell poles. This initial binding facilitates irreversible adsorption to the SPP1 phage receptor protein YueB,which is encoded by a putative type VII secretion system gene cluster.Immunostaining and YueB - GFP fusion showed that the phage receptor protein YueB is found over the entire cell surface. It concentrates at the bacterial poles too,and displays a punctate distribution over the sidewalls. The dynamics of SPP1 DNA entry and replication was visualised in real time by assaying specific binding of a fluorescent protein to tandem sequences present in the SPP1 genome. During infection, most of the infecting phages DNA entered and replicated near the bacterial poles in a defined focus. Therefore, SPP1 assembles a replication factory at a specific location in the host cell cytoplasm. DNA delivery to the cytoplasm depends on millimolar concentrations of Ca2+ allowing uncoupling it from the precedent steps of SPP1 adsorption to the cell envelope and CM depolarization that require only micromolar amounts of this divalent cation. A model describing the early events of bacteriophage SPP1 infection is presented.

Os conhecimentos sobre os mecanismos regulatórios responsáveis pelo crescimento dos fungos filamentosos apresentam lacunas e sua compreensão é necessária para o desenvolvimento de uma terapêutica antifúngica mais adequada, assim como para incrementar a síntese de produtos de interesse comercial. Assim sendo, estudar o envolvimento do Ca2+ na resposta de um fungo modelo como A. nidulans sob fontes de carbono diferentes constitui um meio de gerar conhecimentos sobre as características de crescimento dos fungos filamentosos, de sua resposta a adaptação ambiental e dos mecanismos que controlam essa resposta. Analisou-se na linhagem A26 e na AP27, esta última com ruptura do gene da plcA, o gradiente de Ca2+ citosólico, a morfologia das hifas, a germinação e o ciclo de divisão nuclear quando as linhagens tinham calcineurina ou calmodulina inibidas e quando os canais de Ca2+ estavam bloqueados ou abertos. Os níveis de Ca2+ citosólico na linhagem A26, crescendo em presença de glicose, foram maiores que os detectados em meio suplementado com pectina. O ciclo de germinação e divisão celular no AP27, independentemente da fonte de carbono, mostrou-se mais lento se comparado com a linhagem A26, provavelmente devido ao fato de seus estoques intracelulares de Ca2+, tanto em nível vesicular quanto citosólico, serem menores. A linhagem AP27 apresentou ramificações dicotômicas nas pontas das hifas e nas hifas laterais em ambas as fontes de carbono nas quais foi cultivada, o que não se observou na linhagem A26. Quando calcineurina foi inibida por ciclosporina A, as hifas das duas linhagens, em ambas condições de cultivo, alongaram-se menos e apresentaram-se mais ramificadas, no entanto este efeito foi mais pronunciado em presença de glicose, e entre as duas linhagens pode-se dizer que foi mais intenso na linhagem AP27, demonstrando a importância dos níveis de cálcio na atividade desta enzima e conseqüentemente no desenvolvimento normal das hifas. A abertura dos canais de Ca2+, por ionóforo, produziu hiperramificação em ambas as linhagens, mas principalmente quando cresciam em pectina e ao contrário do efeito observado em presença de verapamil, que bloqueia os canais de Ca2+, não promoveram hifas laterais e nem pontas dicotômicas. No entanto o outro bloqueador dos canais de Ca2+ testado, ácido caurenóico, apresentou efeito morfológico diferente, pois as hifas tornaram-se curvas o que indica perda de polaridade. O inibidor da calmodulina (TFP) retardou a germinação, principalmente no mutante AP27, quando crescendo em presença de glicose. Lembrando que o complexo Ca2+/CaM ativa a calcineurina e que o mutante apresenta menores níveis de cálcio, esse resultado é justificável. A ruptura do gene plcA não impediu o crescimento e desenvolvimento do mutante, provavelmente porque a função desta enzima poder ser provida por outras partes do genoma, mas comprometeu os níveis intracelulares de cálcio e conseqüentemente a sua morfologia. Este estudo mostra a importância da fosfolipase C, para manutenção dos níveis intracelulares de Ca2+, no desenvolvimento normal das hifas de A. nidulans e, pela primeira vez, demonstra que esses níveis são diferentes quando o fungo cresce em presença de uma fonte de carbono, prontamente metabolizável ou não. Esses resultados conferem ao cálcio um papel modulador nessas condições de cultivo. / The knowledge about regulatory mechanisms responsible for filamentous fungi growth presents lacks and its understanding is important to develop adequate antifungal therapy either to contribute the synthesis of interestingly commercial products. By this way, study Ca2+ relationship to a fungal model as A. nidulans about different carbon sources, constitute knowledge about the filamentous fungi growing characteristics, environment adaptation and its control mechanisms. The strains A26 and AP27 was analyzed, this last one with disruption plcA gene, cytosolic Ca2+ gradient hyphal morfology, germination and nuclear division cycle when these strains had calcineurin or calmodulin inhibition and Ca2+ channel were blocked or opened. Cytosolic Ca2+ levels in A26 strain, growing in the presence of glucose was higher than supplemented media with pectin. AP27 strain, independently of carbon source, demonstrated lower germination and cell division than A26 strains, probably due to the fact that intracellular Ca2+ stocks either vesicular as cytosolic levels were lower. AP27 strain presented dichotomous branching at tip-high and lateral hyphae, at both carbon source that was grown, didnt observed at A26 linkage. When calcineurin was inhibited by cyclosporin A, hyphae from both strains, in both growth conditions, had less elongated and showed more branching, however this effect was more pronounced in presence of glucose, and between both strains was more intense at AP27 strain, indicating the importance of Ca2+ levels at this enzymatic activity and therefore the normal development of hyphae. The opening of Ca2+ channel by ionophore, produced hyperbranching in both strains, even when growth in pectin and in contrast of effect observed in the presence of verapamil, that blocks Ca2+ channels, didnt promote lateral or tip high dichotomous branching. However kaurenoic acid, an another Ca2+ channel blocker tested, presented different morphological effect, because hyphae became curved, indicating loss of polarity. Calmodulin inhibitor (TFP) delayed germination mainly at mutant AP27, when growing in the presence of glucose. Remembering that Ca2+/CaM complex activate calcineurin and the mutant exhibit lower Ca2+ levels, justifying this results. The rupture of plcA gene didnt affected growth and development of mutant, probably because the function of this enzyme can be provided by another parts of genoma, damaged the Ca2+ intracellular levels and consequently its morphology. This study shows the importance of fosfolipase C to maintaining the intracellular Ca2+ levels, at the normal hyphae development of A. nidulans and for the first time, demonstrating that this levels are different when fungi are grown in the presence of carbon source, promptly metabolizable or not. This results gives to Ca2+ as modulator at growth conditions.

Les quatre étapes principales d'infection des bactéries par leurs virus sont (i) la reconnaissance spécifique de la cellule hôte et l'entrée du génome dans le cytoplasme,(ii) la réplication du génome viral, (iii) l'assemblage des particules virales, et (iv) leur relâchement, menant dans la plupart des cas à la lyse de la cellule. Bien que la description des étapes individuelles du cycle viral a été relativement bien établie, les détails de comment d'ADN viral chemine du virion jusqu’au cytoplasme de la bactérie hôte et de comment l'environnement cellulaire participe au processus restent mal compris.La première étape de l’infection est la reconnaissance d’un récepteur à la surface de la bactérie hôte par la machinerie d’adsorption du phage. Les barrières que l’agent infectieux doit franchir par la suite sont la membrane externe de la bactérie Gram-negative, la paroi cellulaire et la membrane cytoplasmique. Ceci implique une dégradation localisée de la paroi et le cheminement de l’ADN à travers un pore dans la membrane. L‘ADN linéaire se circularise normalement dans le cytoplasme et il est répliqué par la suite. On a utilisé le bactériophage SPP1 qui infecte la bactérie Gram-positive Bacillus subtilis comme modèle d’étude pour disséquer ces différentes étapes clés pour le démarrage de l’infection virale. Dans ce travail de thèse les conditions d’infection et d’acquisition de données pour suivre en temps réel la dépolarisation de la membrane cellulaire de B. subtilis lors de l’infection par SPP1 ont été mis au point. Il est montré que le démarrage de l’infection déclenche une dépolarisation très rapide de la membrane cytoplasmique.Le potentiel de membrane n’est plus rétablit pendant toute la durée du cycle d'infection. Ce changement du potentiel de membrane au début de l’infection dépend de la présence du récepteur YueB. L’amplitude de la dépolarisation dépend du nombre de particules virales infectieuses présentes et de la concentration du récepteur YueB à la surface de la bactérie hôte. L’interaction du phage avec le récepteur YueB conduit à l’interaction irréversible et à l'éjection de l’ADN de SPP1. Pour établir si c’est l’interaction avec YueB ou le début de l’entrée de l’ADN qui conduit à la dépolarisation de la membrane on a utilisé des phages SPP1 éclates par EDTA qui adsorbent normalement à B. subtilis mais qui n’avaient plus leur ADN. Les résultats obtenus ont montré que la dépolarisation requiert l’interaction du virus intacte avec le récepteur YueB. Des concentrations sous-millimolaire de Ca2+ sont nécessaires et suffisantes pour SPP1 liaison réversible à l'enveloppe d'hôte et donc de déclencher la dépolarisation.La cinétique d’entrée de l’ADN du bactériophage SPP1 dans la bactérie Bacillus subtilis a été suivie en temps réel par microscopie de fluorescence. On a mis au point une méthode de microscopie pour visualiser des particules virales marquées avec des «quantum dots» ce qui permit de démontrer que ces particules se fixent préférentiellement aux pôles des bacilli. L’immuno-marquage du récepteur de SPP1,la protéine YueB, a montré que celle-ci a une organisation ponctuée à la surface de B.subtilis et se concentre particulièrement aux extrémités de la bactérie. Cette localisation particulière du phage sur la surface de la cellule hôte corrèle avec l’observation que l’ADN viral rentre dans le cytoplasme (<2 min) et se réplique dans des foci situés dans la plupart des cas à proximité des pôles de B. subtilis. L’étude spatio-temporelle de l’interaction de SPP1 avec son hôte Gram-positive montre que le virus cible des régions spécifiques de la bactérie pour son entrée et pour sa réplication. Transfert d'ADN dans le cytoplasme dépend des concentrations millimolaires de Ca2+. Un modèle décrivant les événements précoces de l'infection bactériophage SPP1 est présenté. / The four main steps of bacterial viruses (bacteriophages) lytic infection are (i) specific recognition and genome entry into the host bacterium, (ii) replication of the viral genome, (iii) assembly of viral particles, and (iv) their release, leading in most cases to cell lysis. Although the course of individual steps of the viral infection cycle has been relatively well established, the details of how viral DNA transits from the virion to the host cytoplasm and of how the cellular environment catalyzes and possibly organizes the entire process remain poorly understood.Tailed bacteriophages are by far the most abundant viruses that infect Eubacteria. The first event in their infection is recognition of a receptor on the surface of host bacterium by the phage adsorption machinery. The barriers that the infectious particle overcomes subsequently are the cell wall and the cytoplasmic membrane of bacteria. This implies a localized degradation of the wall and the flow of its double stranded DNA (dsDNA) through a hydrophilic pore in the membrane. The lineards DNA molecule is most frequently circularized in the cytoplasm followed by its replication. In this study we used bacteriophage SPP1 that infects the Gram-positive bacterium Bacillus subtilis as a model system to dissect the different steps leading to transfer of the phage genome from the viral capsid to the host cell cytoplasm.normally to B. subtilis but do not trigger depolarization of the CM. Attachment of intact SPP1 particles is thus required for phage-induced depolarization.The beginning of B. subtilis infection by bacteriophage SPP1 was followed inspace and time. The position of SPP1 binding at the cell surface was imaged by fluorescence microscopy using virus particles labeled with "quantum dots". We found that SPP1 reversible adsorption occurs preferentially at the cell poles. This initial binding facilitates irreversible adsorption to the SPP1 phage receptor protein YueB,which is encoded by a putative type VII secretion system gene cluster.Immunostaining and YueB – GFP fusion showed that the phage receptor protein YueB is found over the entire cell surface. It concentrates at the bacterial poles too,and displays a punctate distribution over the sidewalls. The dynamics of SPP1 DNA entry and replication was visualised in real time by assaying specific binding of a fluorescent protein to tandem sequences present in the SPP1 genome. During infection, most of the infecting phages DNA entered and replicated near the bacterial poles in a defined focus. Therefore, SPP1 assembles a replication factory at a specific location in the host cell cytoplasm. DNA delivery to the cytoplasm depends on millimolar concentrations of Ca2+ allowing uncoupling it from the precedent steps of SPP1 adsorption to the cell envelope and CM depolarization that require only micromolar amounts of this divalent cation. A model describing the early events of bacteriophage SPP1 infection is presented.