Submitted by Luiz Felipe Barbosa (luiz.fbabreu2@ufpe.br) on 2015-03-13T13:23:37Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese - Rodrigo Mendonça de Lucena.pdf: 1925141 bytes, checksum: ad20740a309b4280513e7c3cc2a874b1 (MD5) / Made available in DSpace on 2015-03-13T13:23:37Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese - Rodrigo Mendonça de Lucena.pdf: 1925141 bytes, checksum: ad20740a309b4280513e7c3cc2a874b1 (MD5) Previous issue date: 2012 / FACEPE / Células de S. cerevisiae são submetidas a diferentes tipos de estresse durante o processo fermentativo para a produção de álcool combustível. No Brasil, este processo ocorre com reciclos celulares intercalados por etapas de pré-fermentação que inclui, entre outros procedimentos, o tratamento com ácido sulfúrico diluído para controle da população bacteriana. Este tratamento conduz a perda da viabilidade celular, com conseqüências no rendimento fermentativo. Considerando estes fatos, o presente trabalho tem o propósito de identificar a resposta genética e metabólica de S. cerevisiae ao estresse ácido, e revelar os mecanismos que conduzem a tolerância e adaptação celular. Foi realizada a triagem de mutantes com deleções crescendo em meio com pH neutro e ácido, onde foram comparados com a linhagem selvagem, assim como também foi avaliada a expressão gênica global e específica para genes envolvidos em diferentes via metabólicas. Os resultados mostraram que a via de Integridade da Parede Celular é o principal mecanismo responsável pela tolerância celular ao pH ácido, onde este dano ativa a via da proteína quinase C (PKC) principalmente pelo sensor de membrana Wsc1p. Adicionalmente, danos a parede celular podem mimetizar o efeito do choque hiperosmótico e ativar a via HOG, a qual amplifica o sinal da via PKC e induz a ativação dos canais de Ca2+ pelo aumento da expressão do gene SLT2, promovendo o influxo de cálcio que ativará a calcineurina. Juntos, estes mecanismos conduzem a mudanças na expressão de genes envolvidos com a regeneração da parede celular, metabolismo de carboidratos, resposta a feromônios e regulação do ciclo celular.

Submitted by Caroline Falcao (caroline.rfalcao@ufpe.br) on 2017-04-10T17:50:09Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 2012-Tese-RodrigoLucena.pdf: 1924564 bytes, checksum: 5c075accad4cf839a0292842848e65e3 (MD5) / Made available in DSpace on 2017-04-10T17:50:09Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 2012-Tese-RodrigoLucena.pdf: 1924564 bytes, checksum: 5c075accad4cf839a0292842848e65e3 (MD5) Previous issue date: 2012 / Células de S. cerevisiae são submetidas a diferentes tipos de estresse durante o processo fermentativo para a produção de álcool combustível. No Brasil, este processo ocorre com reciclos celulares intercalados por etapas de pré-fermentação que inclui, entre outros procedimentos, o tratamento com ácido sulfúrico diluído para controle da população bacteriana. Este tratamento conduz a perda da viabilidade celular, com conseqüências no rendimento fermentativo. Considerando estes fatos, o presente trabalho tem o propósito de identificar a resposta genética e metabólica de S. cerevisiae ao estresse ácido, e revelar os mecanismos que conduzem a tolerância e adaptação celular. Foi realizada a triagem de mutantes com deleções crescendo em meio com pH neutro e ácido, onde foram comparados com a linhagem selvagem, assim como também foi avaliada a expressão gênica global e específica para genes envolvidos em diferentes via metabólicas. Os resultados mostraram que a via de Integridade da Parede Celular é o principal mecanismo responsável pela tolerância celular ao pH ácido, onde este dano ativa a via da proteína quinase C (PKC) principalmente pelo sensor de membrana Wsc1p. Adicionalmente, danos a parede celular podem mimetizar o efeito do choque hiperosmótico e ativar a via HOG, a qual amplifica o sinal da via PKC e induz a ativação dos canais de Ca2+ pelo aumento da expressão do gene SLT2, promovendo o influxo de cálcio que ativará a calcineurina. Juntos, estes mecanismos conduzem a mudanças na expressão de genes envolvidos com a regeneração da parede celular, metabolismo de carboidratos, resposta a feromônios e regulação do ciclo celular. / S. cerevisiae cells are subjected to different sorts of stress during the fermentation process for the fuel alcohol production. In Brazil, this process occurs using cell recyclings interspersed with steps of pre-fermentation which includes, among other procedures, treatment with dilute sulfuric acid to control of the bacterial population. This treatment leads to loss of cell viability, with consequences on yield fermentation. Considering these facts, the present study aims to identify the genetic and metabolic response of S. cerevisiae during acid stress, and reveal the mechanisms leading to tolerance and cellular adaptation. Screening was performed with deletion mutants grown in media with neutral and acid pH, which were compared with the wild strain, as well as also was evaluated the global and specififies gene expression to genes involved in different metabolic pathway. The results showed that the Cell Wall Integrity pathway is the main mechanism responsible for cellular tolerance to acid pH, where the damage activates the protein kinase C (PKC) mainly by Wsc1p membrane sensor. In addition, cell wall injury might mimic the effects of high osmotic shock and activates the HOG pathway, which amplifies the signal in the upper part of PKC pathway and leads to the activation of Ca2+ channels by SLT2 overexpression and this Ca2+ influx further activates calcineurin.Together, these mechanisms induce the expression of genes involved in cell wall regeneration, mating, carbohydrate metabolism and cell cycle regulation.

In the yeast Saccharomyces cerevisiae, stress on the cell wall is caused by various external influences (e.g. exposure to chemicals, oxidative stress, osmotic changes, pH changes or heat shock) which trigger the cell wall integrity signalling pathway (CWI). The aim of my work was to investigate the effect of the CWI pathway on yeast colony morphogenesis. Using strains with deletions in genes of the CWI pathway derived from two parental strains BR-F-Flo11p-GFP and PORT, I have found that differences in genetic background influences the process and activation of this pathway. Among the strains derived from BR-F-Flo11p-GFP, only the strain with the deletion of MID2 affects the appearance of colonies. MID2 encodes a cell-surface sensor of CWI pathway. In all deletion strains derived from PORT, the disruption of the CWI pathway causes a slower development of colonies growing on glycerol medium supplemented with 0,05 mM selenate inducing fluffy colony morphology. The largest effect has deletion of gene MTL1 which also encodes a cell-surface sensor with homology to Mid2. I have confirmed that strains with deletions in genes of CWI pathway have altered sensitivity to inhibitors disrupting cell wall integrity (Calcofluor white, Congo red, zymolyase). By means of zymolyase assay, I have confirmed the...

La paroi de la levure Saccharomyces cerevisiae est une structure très dynamique composée de beta-glucanes, de mannanes et de chitine (polymère de N-acétylglucosamine). Elle peut s’adapter à l’état physiologique et aux changements morphologiques des cellules, ainsi qu’aux contraintes environnementales. Cette remarquable plasticité est assurée par l’intervention de différentes voies de régulation et de signalisation dont la voie CWI (Cell Wall Integrity) et la voie de la Calcineurine ou Protein Phosphatase 2B. La toxine killer K9 est une petite protéine sécrétée par la levure Hansenula mrakii. Cette toxine exerce son action létale sur les souches contrôles de S. cerevisiae mais pas sur des mutants du gène KNR4. Elle inhibe in vitro la beta-(1,3)-glucan syntase. Ce travail a dans un premier temps utilisé la Microscope à Force Atomique (AFM) et mis en évidence que la paroi de S. cerevisiae contrôle et mutant knr4 sont affectées de façon similaires par un traitement par la toxine K9. Dans un second temps, nous avons pu démontrer que la localisation cellulaire de Knr4 aux sites de croissance polarisée est nécéssaire pour l’action létale de la toxine K9 sur les cellules de S. cerevisiae.Knr4 fait partie d’une famille de protéines très conservées dans le domaine fongique, impliquées dans le contrôle de l’intégrité pariétale et la morphogenèse. Elle constitue un élément coordinateur pour la voie CWI et la voie de la Calcineurine. Notre travail a mis en évidence que la phosphorylation des résidus serine 200 et serine 203 de Knr4 joue un rôle dans ce mécanisme de coordination / The aim of my thesis was to study the fuction of Knr4 in the cell wall synthesis, morphogenesis, and related signaling pathways. The content of my thesis is mainly divided into three parts: The first part concerns our search to find out unknown partners of Knr4 and to investigate the cellular pathways required for localization of Knr4 protein. To that end, we decided to use a series of deletion mutants interrupted in genes related to morphogenesis and establishment of cellular polarity. We selected candidate genes from the Saccharomyces cerevisiae genome database (SGD, Stanford), using the keywords “Morphogenesis” and “Cell Polarity”. After selection and addition, 25 genes related to the morphogenesis and cell polarity were chosen for our Knr4 localization analysis. Through analysis of the results, we got 10 interesting mutants related to morphogenesis and polarity in which knr4 protein localization was affected: bem2Δ, pcl1Δ, pcl2Δ, rrd1Δ, spa2Δ, tpd3Δ, bem1Δ, bnI1Δ, yck1Δ and bud6Δ, and two additional mutants pph21Δ related to the tpd3Δ and cna1Δ involved in the calcinerin pathway. The second part deals with a mutational analysis of in vivo phosphorylated residues of Knr4 in the function and localization the protein, as well as in the modulation of calcineurin activity and CWI pathway. We found that S200S203 phosphorylation mutants cannot rescue viability of a double mutant bck1Δknr4Δ, while they can rescue slt2Δknr4Δ. In addition, S200S203 phosphorylation mutants behave as the absence of Knr4 towards suppression of lethality caused by an hyperactivated Mkk1 allele. Also we found that the knr4with KNR4S200AS203A mutant can results in hyperactivation of the Calcineurin pathway compared to control situation. So serin 200 and serin 203 may be involved in the cross-talking with the calcineurin pathway and CWI pathway. The third part is the study of K9 killer toxin’s strong cytocidal activity against sensitive yeast strains, including Saccharomyces cerevisiae. Treatment with this toxin results in the formation of pores at the surface of the cells, and more specifically at places where cell wall synthesis is the most active, namely at the tip of growing buds or mating projections. Yeast cells treated with K9 toxin then die by releasing cytoplasm and cellular materials from these pores. In the yeast S. cerevisiae, Knr4 protein localizes at the sites of polarized growth (bud tips, shmoo tips), which are also the sites where the toxin forms pores in the cell wall. Mutants defective in KNR4 gene are remarkably resistant to this toxin. In this study, we analyzed for the first time the biophysical effects of K9 on the yeast cell wall using Atomic Force Microscopy (AFM), a cutting edge technology that allows measuring the nanomechanical properties of living yeast cells, and their alterations by various drugs. To this end, we measured the effects of K9 toxin on the nanomechanical properties of the cell wall of S. cerevisiae wild-type cells and mutants deleted for KNR4 gene, at the short (2 h) and long term (20 h). Our results reveal an important cell wall remodeling occurring in wild-type cells already after 2 hours and only visible in knr4 mutant after 20 hours of treatment. Moreover, we investigated the role of Knr4 protein in the cells sensitivity towards the toxin. We were able to show that the presence of the N-terminal domain of Knr4 protein, which is required for its correct cellular localization at the bud tip during cell cycle, is essential for the toxin K9 wild-type sensitivity. In addition, a series of deletion mutants from the YKO collection in which the Knr4 cellular localization is also lost display a reduced sensitivity to the K9 toxin. Taken together, these results shed light on the importance of the proper localization of Knr4 protein at sites of intensive cell wall growth for the wild-type cells sensitivity to K9 killer toxin.

Dans l'optique de produire des agro‐carburants, le sorgho sucré est aujourd'hui proposé comme une alternative à d'autres espèces cultivées à grande échelle comme la canne à sucre et le maïs car il présente plusieurs avantages : le sorgho est résistant à la sécheresse et à la chaleur, il nécessite peu d'intrants, a en moyenne un cycle de culture relativement court (3‐4 mois) comparé à la canne à sucre. Il offre une grande diversité génétique à explorer et exploiter, tout en étant génétiquement moins complexe que la canne à sucre. Finalement, il peut être cultivé pour un double usage, le grain pouvant être utilisé comme source d'alimentation pour l'homme ou le bétail (à partir du grain) et le jus sucré contenu par les tiges comme source d'agrocarburant. Cette polyvalence en fait une culture idéale pour lutter contre la compétition entre cultures énergétiques et cultures vivrières et assurer des rendements dans des environnements de culture sujets au stress hydrique et thermique comme c'est le cas en Afrique de l'Ouest. Cependant, le caractère sucré du sorgho est complexe, car sous l'influence d'interactions Génotype X Environnement (GxE). Aussi, les mécanismes métaboliques, morphologiques ou phénologiques constituant la cinétique d'accumulation des glucides dans la tige et son éventuelle compétition avec le remplissage des grains restent mal connus ou très controversés dans la littérature. La présente thèse, réalisée dans le cadre du projet européen Sweetfuel, vise à comprendre ces mécanismes, afin de contribuer à la définition d'idéotypes de sorgho double usage, pour les environnements soudano‐sahéliens.Sur la base d'études expérimentales au champ au Mali et en serre en France, il a pu être démontré que les glucides sont accumulés dans les entrenoeuds des tiges par un jeu d'activités enzymatiques (favorisant l'accumulation d'hexoses puis de saccharose) dès le début de leur élongation, donc potentiellement avant la floraison. Au Mali, l'étude au champ de 14 génotypes adaptés aux conditions locales, plus ou moins sensibles à la photopériode et semés à trois dates différentes, a démontré le bénéfice d'un rallongement de la phase végétative sur la quantité de sucre accumulée dans les tiges de la plante à floraison, du fait d'un plus grand nombre d'entrenoeuds allongés et du temps à leur disposition pour accumuler des glucides avant ce stade. Ce bénéfice était cependant plus lié à la plus grande quantité de biomasse accumulée (taille des tiges) qu'à la concentration en sucre dans les entrenoeuds (plutôt stable entre dates de semis).Ainsi, la durée de la phase végétative et la sensibilité à la photopériode sont proposés comme des paramètres clés favorisant la quantité de glucides accumulée dans les tiges de la plante à floraison. D'autre part, il a été montré que la quantité de glucides présente à maturité dans les tiges des mêmes génotypes ne différait pas ou peu de celle à floraison, une éventuelle réduction pour quelques génotypes n'étant généralement pas significative et évitable par l'allongement du cycle. De plus, cette quantité de glucides dans les tiges à maturité n'a tiré aucun bénéfice de l'ablation de la panicule à floraison chez les mêmes génotypes. Ces résultats suggèrent que la compétition entre le remplissage du grain et la production de sucre est faible chez le sorgho, d'autant plus faible que la plante présente de grandes tiges et donc un grand compartiment de stockage des glucides, tamponnant cette éventuelle compétition. D'ailleurs, à une échelle plus fine, aucune différence n'a pu être mise en évidence en termes d'activité des principales enzymes du métabolisme carboné dans la tige d'un génotype dans sa version stérile (pas de remplissage du grain) et fertile.Ce travail a démontré le potentiel du sorgho pour une double utilisation dans un contexte soudano‐sahélien et la pertinence d'exploiter la diversité génétique de cette espèce pour cette objectif de sélection. Les résultats ob / Sweet sorghum offers many advantages as an alternative to widely cultivated crops such as corn and sugarcane to produce biofuels: it is resistant to water stress, it requires few inputs; it has a shorter growth cycle compared to sugarcane in particular. Sorghum also exhibits a great genetic diversity and is genetically less complex than sugarcane. Finally, sorghum can be cultivated for dual‐purpose uses, using grains for food or feed and sweet juice for biofuel production. Hence, sorghum is a promising option to reduce the competition for land and (water) resource use between food and fuel, in particular in cropping environments with high drought and heat stress frequency, as in West Africa. However, stem sweetness is a complex trait prone to genotype x environment interactions (GxE). The metabolic, morphological and phenological mechanisms involved in the kinetic of stem sugar accumulation and its possible competition with grain filling are largely unknown or controversial in the literature. The present work is part of the European project Sweetfuel and aims at better understanding these mechanisms and contributing to define dual‐purpose sorghum ideotypes for soudano‐sahelian conditions.Based on field and greenhouse experiments respectively in Mali and France, it was found that sugars start accumulating in stem internodes at the onset of their elongation, i.e. potentially soon before the plant flowers. The successive accumulation of hexose and then sucrose in internodes could be dynamically explained by changes in the activity of key enzymes related to sucrose metabolism. In Mali, a field experiment performed on 14 genotypes, contrasted for photoperiod sensitivity and sown at three planting dates, highlighted the interest of increasing vegetative phase duration to increase sugar yield. This was explained first of all by the higher number of internodes that could expand during a longer vegetative phase, and thus, by the higher production of stem biomass, and, to a minor extent, by the longer time for internodes to mature and accumulate sugar (sugar concentration in the stem was however fairly stable across sowing dates). Also, vegetative phase duration and photoperiod sensitivity can be considered as two key parameters promoting stem sugar content before grain filling. In the same time, it was shown that stem sugar content kept remarkably constant between anthesis and maturity in most of studied genotypes and that the reduction observed for some genotypes was overcome with an early sowing. Moreover, sugar accumulation in the stem between flowering and maturity did not benefit from panicle pruning. These results together suggest that the competition for carbohydrates between stem sugar reserves and grain filling is weak; it is even weaker for big/large stem genotypes with huge sugar reserves in the stem that would buffer a post‐flowering allocation of sugar from the stem to the grains if required. This low competition was confirmed at a finer scale, as no differences were observed in the activity of key enzymes of sucrose metabolism between the sterile and the fertile line of a same genotype.This work demonstrates the potential of sorghum for dual‐purpose in particular for soudano‐sahelian cropping conditions and the interest of using its genetic diversity for this breeding purpose. It provides further knowledge for revisiting the phenotyping strategies to be adopted to investigate the genetic basis of sugar and grain production and their combination. The results are also currently used to improve the way the source‐sink relationships underlying this dual production are formalized in crop and plant models at CIRAD. Such models will be then useful to assist sorghum ideotype exploration for dual purpose.

La coda ultrasonore présente une haute sensibilité aux perturbations du milieu de propagation. L'analyse de la coda par la Coda Wave Interferometry (CWI) permet d'évaluer précisément la variation de la vitesse de propagation (résolution en relatif de 0,001 %). Une telle évaluation peut être utilisée pour le contrôle non destructif (ECND) d’un matériau ou d'une structure.Un essai expérimental est présenté au début comme exemple d’utilisation de la CWI pour l’ECND du béton. Face aux problèmes inhérents au degré de précision de cet essai, nous présentons un protocole conçu pour améliorer la fiabilité des résultats CWI. Nous vérifions expérimenta vérifient que ce protocole peut améliorer la répétabilité de l’essai en réduisant les biais provenant des fluctuations de température ambiante et des procédures expérimentales. Avec ce protocole, une étude du comportement de béton sous un chargement uni-axial en traction directe a été effectuée en utilisant la CWI. Les comportements élastique (l’effet acoustoélastique) et anélastique (l’effet Kaiser) du béton sont observés via les résultats CWI. Un coefficient acoustoélastique effectif (Bêta) a été déterminé et utilisé pour la détection d’un endommagement léger du béton.La CWI est ensuite utilisée pour la détection globale de défauts dans un milieu initialement linéaire (verre) en observant la modulation non linéaire. L’apparition de ce phénomène non linéaire est due à la présence des défauts et détectée par la variation des résultats CWI en fonction de l’amplitude de l’onde de pompe. Nous montrons que cette méthode permet la détection des défauts et d’évaluation du niveau d’endommagement d’une manière globale sans zone aveugle. / With their long and complex propagation paths, coda waves can probe the propagation medium repeatedly and show a high sensitivity to the perturbations to the medium, i.e. variations in propagation velocity. Since such variations may indicate the modification of elastic properties and Coda Wave interferometry (CWI) can determine it precisely (relative resolution of 0.001%), CWI is considered a promising method for non destructive testing and evaluation (NDT&E). An experimental test is presented as an example of the CWI use on concrete for NDT&E purpose. For solving the experimental repeatability issue revealed in this test, a bias-control protocol is designed to reduce the experimental bias in CWI results. It is experimentally confirmed that this protocol can remarkably improve the reliability of CWI results and the experimental repeatability. Together with this bias-control protocol, the CWI is used to study the behaviors of concrete under uni-axial load in direct tension. Both elastic (acoustoealstic effect) and inelastic (Kaiser effect) behaviors are observed via CWI results. Effective value of acoustoelastic coefficient is then determined from CWI results and used for the detection of an early-stage damage that artificially induced to the concrete specimen. A defect-detection method of an initially linear medium (glass) is then developed by using the CWI. Due to the nonlinearity brought by the defects, acoustic mixing effect occurred, and the observation of such effect is the indication of damage. The use of a broadband pump wave and the CWI makes possible to 1) detect the damage globally without blind zone and 2) assess the damage level in an effective manner.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第18326号 / 農博第2051号 / 新制||農||1022(附属図書館) / 学位論文||H26||N4833(農学部図書室) / 31184 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 植田 充美, 教授 喜多 恵子, 教授 栗原 達夫 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

As the number of Indigenous children and youth in the care of Manitoba child welfare steadily increases, so do the questions and public debates. The loss of children from Indigenous communities due to residential schools and later on, to child welfare, has been occurring for well over a century and Indigenous people have been continuously grieving and protesting this forced removal of their children. In 1999, when the Manitoba government announced their intention to work with Indigenous peoples to expand off-reserve child welfare jurisdiction for First Nations, establish a provincial Métis mandate and restructure the existing child care system through legislative and other changes, Indigenous people across the province celebrated it as an opportunity for meaningful change for families and communities. The restructuring was to be accomplished through the Aboriginal Justice Initiative-Child Welfare Initiative (AJI-CWI). Undoubtedly, more than a decade later, many changes have been made to the child welfare system but children are still been taken into care at even higher rates than before the changes brought about by the AJI-CWI. In order to develop an understanding of what has occurred as a result of the AJI-CWI process, this study reached out to child welfare workers who had worked in the system before, during and after the process was put in place. Using a storytelling approach based in an Indigenous methodology, twenty-seven child welfare workers shared how they perceived the benefits, the deficits, the need for improvement and how they observed the role of Indigenous culture within the child welfare context. The stories provide a unique insight into how the changes were implemented and how the storytellers experienced the process, as well as their insights into barriers, disappointments, benefits and recommendations for systemic change. / Graduate

La coda ultrasonore présente une haute sensibilité aux perturbations du milieu de propagation. L'analyse de la coda par la Coda Wave Interferometry (CWI) permet d'évaluer précisément la variation de la vitesse de propagation (résolution en relatif de 0,001 %). Une telle évaluation peut être utilisée pour le contrôle non destructif (ECND) d'un matériau ou d'une structure.Un essai expérimental est présenté au début comme exemple d'utilisation de la CWI pour l'ECND du béton. Face aux problèmes inhérents au degré de précision de cet essai, nous présentons un protocole conçu pour améliorer la fiabilité des résultats CWI. Nous vérifions expérimenta vérifient que ce protocole peut améliorer la répétabilité de l'essai en réduisant les biais provenant des fluctuations de température ambiante et des procédures expérimentales. Avec ce protocole, une étude du comportement de béton sous un chargement uni-axial en traction directe a été effectuée en utilisant la CWI. Les comportements élastique (l'effet acoustoélastique) et anélastique (l'effet Kaiser) du béton sont observés via les résultats CWI. Un coefficient acoustoélastique effectif (Bêta) a été déterminé et utilisé pour la détection d'un endommagement léger du béton.La CWI est ensuite utilisée pour la détection globale de défauts dans un milieu initialement linéaire (verre) en observant la modulation non linéaire. L'apparition de ce phénomène non linéaire est due à la présence des défauts et détectée par la variation des résultats CWI en fonction de l'amplitude de l'onde de pompe. Nous montrons que cette méthode permet la détection des défauts et d'évaluation du niveau d'endommagement d'une manière globale sans zone aveugle.