La reprogrammation du métabolisme est maintenant considérée comme des caractéristiques des cellules cancéreuses et une conséquence de leur adaptation à un microenvironnement hostile se traduisant par une baisse de la concentration d’oxygène et de la disponibilité des nutriments. Donc, les cellules cancéreuses sont capables d’adapter leur métabolisme pour survivre et proliférer. Des avancées récentes dans la connaissance de ces modifications permettent l’émergence de nouvelles approches thérapeutiques ciblant spécifiquement ces changements métaboliques. Un des principaux régulateurs du métabolisme cellulaire est le coactivateur transcriptionnel PGC-1α (PPARgamma coactivator1-alpha). PGC-1α contrôle, entre autres, la biogénèse mitochondriale, la phosphorylation oxydative et l’oxydation des acides gras. Récemment, il a été montré que PGC-1α facilite la biogénèse mitochondriale dans les cellules cancéreuses du sein et augmentent significativement leurs potentiels métastatiques. Au contraire, il a été montré que la surexpression de PGC-1α diminue la formation de métastases dans le mélanome et l’adénocarcinome prostatique. Cependant, les modifications métaboliques et moléculaires conduisant à l’agressivité du cancer de la prostate sont, à l’heure actuelle, peu connues. Dans ce contexte, le but de ma thèse était d’étudier le rôle de PGC-1α sur le métabolisme et l’agressivité des cellules cancéreuses de prostate. Au cours de ma thèse, nous avons démontré que la diminution de l’expression de PGC-1α augmente les trois caractéristiques fondamentales de l’agressivité tumorale : la prolifération, la migration et l’invasion. Afin de déterminer les modifications métaboliques impliquées dans ce phénotype, nous avons réalisé des expériences de métabolomiques en comparant les cellules contrôles aux cellules dont l’expression de PGC-1α est diminuée (shPGC-1α). Nous avons montré que la baisse de PGC-1α augmente significativement la biosynthèse des polyamines. Les polyamines sont impliquées dans de nombreuses fonctions cellulaires, en particulier la prolifération et la migration cellulaire. Ainsi, nous avons inhibé la synthèse des polyamines avec le DFMO, l’inhibiteur de l’enzyme limitante de la voie : ODC, ou bien des siRNA dirigés contre ODC. Nous avons montré que les effets pro-migratoires et pro-invasifs dus à l’invalidation de PGC-1α sont bloqués par le DFMO et les siRNA ODC. De façon intéressante, l’ajout de polyamines exogènes restaure partiellement l’agressivité des cellules. En accord avec ces résultats, nous montrons que ODC est surexprimée quand PGC-1α est diminué et que l’expression de ODC est régulée positivement par l’oncogène c-MYC. En s’intéressant plus en détail à cet oncogène, nous observons que son niveau d’expression augmente dans les cellules invalidées pour PGC-1α et que l’inhibition de c-MYC bloque les effets pro-migratoires et pro-invasifs dus à l’invalidation de PGC-1α. Donc c-MYC participe au phénotype agressif lié à l’augmentation de la voie de biosynthèse des polyamines. Ces résultats in vitro ont été confirmés in vivo par l’analyse des micro-métastases, ils démontrent que les cellules shPGC-1α forment plus de métastases et le traitement par le DFMO inhibe la formation de micro-métastases. Finalement, les données cliniques démontrent que l’expression de PGC-1α est diminuée chez des patients atteints de cancer de la prostate, et cette diminution est corrélée avec une augmentation de c-MYC et ODC. En conclusion, nous avons démontré que PGC-1α est le régulateur majeur d’une voie onco-métabolique par c-MYC et qui promeut l’agressivité du cancer de la prostate par l’intermédiaire de la voie de biosynthèse des polyamines. Ce nouveau circuit métabolique représente une cible thérapeutique intéressante pouvant aider à freiner les formes avancées du cancer de la prostate. / Metabolism reprogramming are now considered to be characteristic of cancer cells and a consequence of their adaptations to a hostile microenvironment resulting in a decrease in oxygen concentration (hypoxia) and the availability of nutrients, particularly glucose and glutamine. Thus, cancer cells can adapt their metabolism to survive and proliferate. Recent advances in the knowledge of these modifications allow the emergence of new therapeutic approaches targeting these metabolic changes. One of the main regulators of cellular metabolism is the transcriptional coactivator PGC-1α (peroxisome proliferator-activated receptor gamma coactivator 1-alpha). PGC-1α controls mitochondrial biogenesis, oxidative phosphorylation and fatty acid oxidation. Recently, it has been shown that PGC-1α promotes mitochondrial biogenesis in cancer cells and dramatically increases their metastatic potential. On the contrary, it appears that overexpression of PGC-1α decreases the formation of metastases in melanoma and prostatic adenocarcinoma. However, the metabolic and molecular changes leading to the aggressiveness of prostate cancer are unclear. Oncogenes and tumor suppressor genes are known to be able to regulate the metabolic adaptations of cancer cells. Several studies show that the number of copies of the gene is increased in 30% of prostate cancers. Transgenic mice overexpressing c-MYC in the prostate develop prostatic intraepithelial neoplasia followed by prostatic adenocarcinoma. At the cellular level, c-MYC expression has been shown to stimulate glycolysis and glutaminolysis in tumor cells, by controlling the expression of genes involved in these metabolic pathways. In addition, c-MYC is also able to increase the polyamine synthesis pathway by inducing the expression of the limiting enzyme of this pathway, ornithine decarboxylase (ODC). In this context, the purpose of my thesis was to study the role of PGC-1α on the metabolism and aggressiveness of prostate cancer cells. During my thesis, we have shown that the decrease of PGC-1α expression increases the three fundamental characteristics of tumor aggressiveness: proliferation, migration and invasion. To determine the metabolic changes involved in this phenotype, we performed metabolic experiments and compared control cells to cells where PGC-1α expression is decreased. We show that the decrease of PGC-1α significantly increases the biosynthesis of polyamines. Polyamines are involved in many cellular functions, particularly in proliferation and cell migration. Thus, we inhibit the synthesis of polyamines with DFMO, an inhibitor ODC which is the rate limiting enzyme of this pathway. We have shown that pro-migratory and pro-invasive effects due to PGC-1α knockout are blocked by DFMO and ODC siRNA. Interestingly, the addition of exogenous polyamine partially restores the aggressiveness of the cells. Consistent with these results, we show that ODC is overexpressed when PGC-1α is decreased and that ODC expression is upregulated by the c-MYC oncogene. In addition, we observe that c-MYC expression increases in cells invalidated for PGC-1α and that the inhibition of c-MYC blocks the pro-migratory and pro-invasive effects due to the invalidation of PGC-1α. Therefore, c-MYC participates in the aggressive phenotype related to the increase of the polyamine biosynthesis pathway. These in vitro results were confirmed in vivo by micro-metastasis analysis, we demonstrate that shPGC-1α cells form more metastases and treatment with DFMO inhibits the formation of micro-metastases. Clinical data indicate that PGC-1α expression is decreased in patients with prostate cancer, and this decrease correlates with an increase in c-MYC and ODC. In conclusion, we show that PGC-1α is the major regulator of an onco-metabolic which promotes prostate cancer aggressiveness via the polyamine biosynthesis pathway.

Orientador: Elizabeth Orika Ono / Resumo: O tomateiro é uma hortaliça de importância econômica e sua produção está relacionada, principalmente, à utilização de produtos químicos, podendo ser reguladores vegetais como as poliaminas e nesse estudo, foi avaliada a ação da putrescina (Put). Assim, o objetivo deste trabalho foi avaliar as respostas fisiológicas do tomateiro à putrescina na redução dos efeitos de estresse hídrico. Para tanto, foram conduzidos dois experimentos em delineamento inteiramente ao acaso (DIC) em esquema fatorial 2 x 4, plantas com e sem deficiência hídrica e quatro doses de putrescina (Put 0, 4, 8 e 12μM), com quatro repetições cada. As doses foram aplicadas via foliar na cultura no momento de submissão do tomateiro ao estresse hídrico, sendo esse no terceiro racemo de flores de cada experimento. O efeito das doses do produto foi avaliado por análises biométricas, bioquímicas de folhas, produtivas e físico/químicas dos frutos pós-colheita. Os resultados obtidos apresentaram maior atividade da putrescina na dose de 12 μM em plantas nos dois experimentos realizados. / Abstract: The tomato is a vegetable of economic importance and its production is available, mainly, to the use of chemical products, which can be plant regulators such as polyamines and, in this study, it was evaluated in a putrescine action (Put). Thus, the objective of this work was to evaluate the borrower's physiological responses to putrescine in reducing the effects of water stress. For this purpose, two experiments were conducted in outdoor design (DIC) in a 2 x 4 factorial scheme, plants with and without hydric and four doses of putrescine (Put 0, 4, 8 and 12μM), with four replicates each. As the doses were applied via leaf culture at the time of the borrower's submission to water stress, this being the third flower motif of each experiment. The effect of product doses was evaluated by biometric, biochemical leaf, productive and physical / chemical analysis of post-harvest fruits. The results obtained showed greater putrescine activity at a dose of 12 μM in plants in the two experiments performed. / Mestre

Malaria is prevalent in over 100 countries which is populated by half of the world’s population and culminates in approximately one million deaths per annum, 85% of which occurs in sub-Saharan Africa. The combined resistance of the mosquitoes and parasites to the currently available pesticides and antimalarial chemotherapeutic agents requires the concerted effort of scientists in the malaria field to identify and develop novel mechanisms to curb this deadly disease. In this study, a thorough understanding of the role players in the polyamine pathway of the parasite was obtained, which could aid future studies in the development of novel inhibitory compounds against these validated drug targets. The uniquely bifunctional S-adenosylmethionine decarboxylase/ornithine decarboxylase (AdoMetDC/ODC) of Plasmodium falciparum forms an important controlling node between the polyamine and methionine metabolic pathways. It has been speculated that the unique bifunctional association of the rate-limiting enzymes allows for the concerted regulation of the respective enzyme activities resulting in polyamine synthesis as per requirement for the rapidly proliferating parasite while the methionine levels are strictly controlled for their role in the methylation status. The results of this study showed that the enzyme activities of the bifunctional complex are indeed coordinated and subtle conformational changes induced by complex formation is suggested to result in these altered kinetics of the individual AdoMetDC and ODC domains. Studies also showed that the identification of the interaction sites between the domains, which allows for communication across the complex, may be targeted for specific interference with the enzyme activities. Furthermore, these studies showed that the current knowledge on the different subclasses of the AdoMetDC family should be re-evaluated since P. falciparum AdoMetDC shows diverse properties from orthologues and therefore points towards a novel grouping of the plasmodial protein. The extensive biochemical and biophysical studies on AdoMetDC has also provided important avenues for the crystallisation and solving of this protein’s 3D structure for subsequent structure-based identification of drug-like lead compounds against AdoMetDC activity. The application of structure-based drug design on malarial proteins was additionally investigated and consequently proved that the rational design of lead inhibitory compounds can provide important scaffold structures for the identification of the key aspects that are required for the successful inhibition of a specific drug target. Spermidine synthase, with its intricate catalytic mechanism involving two substrate binding sites for the products of the reactions catalysed by AdoMetDC/ODC, was used to computationally identify compounds that could bind within its active site. Subsequent testing of the compounds identified with a dynamic receptor-based pharmacophore model showed promising inhibitory results on both recombinant protein and in vitro parasite levels. The confirmation of the predicted interaction sites and identification of aspects to improve inhibitor interaction was subsequently investigated at atomic resolution with X-ray protein crystallography. The outcome of this doctoral study shows the benefit in applying a multidisciplinary and multinational approach for studying drug targets within the malaria parasite, which has led to a thorough understanding of the targets on both biochemical and structural levels for future drug design studies. / Thesis (PhD)--University of Pretoria, 2011. / Biochemistry / unrestricted

Tesis por compendio / [ES] En la presente tesis doctoral se ha evaluado el uso de nuevos soportes celulósicos y silíceos como sistemas de filtración para la estabilización y conservación de alimentos líquidos con el fin de afrontar dos grandes retos de la industria de bebidas. Por un lado, evitar o minimizar los cambios en las propiedades nutricionales, estructurales y organolépticas de los alimentos, ocasionados por la pasteurización térmica tradicional, y ofrecer una alternativa al problema de la baja viabilidad debida a los altos costos de inversión/producción al aplicar nuevas tecnologías no térmicas. Por ello, esta tesis doctoral se centra en el desarrollo y evaluación de una nueva tecnología no térmica de conservación de alimentos líquidos basada en la filtración. Se han desarrollado sistemas de filtración a partir de soportes celulósicos y silíceos, sin funcionalizar o funcionalizados con compuestos antimicrobianos. En el primer capítulo se evaluó el uso de materiales de celulosa como soportes filtrantes para el tratamiento de alimentos líquidos. Como primera aproximación se desarrolló un material poroso nano-micro tubular a partir de la extracción y deslignificación del material celulósico presente en el corazón o raquis de la mazorca de maíz. El uso de este soporte resultó ser efectivo como material filtrante para el tratamiento de agua y zumo de naranja, en un sistema de flujo continuo, eliminando la carga microbiana. La aplicación de este soporte como sistema de filtración presenta diversas ventajas como su capacidad de retención microbiana, la reutilización de sub-productos del maíz y, por tanto, su respeto al medioambiente. Sin embargo, sería necesario optimizar el proceso de filtrado para evitar la frecuente obturación de sus poros que requirió varios ciclos de lavado durante el proceso, así como establecer un método de regeneración del material para incrementar su vida útil. Además, este sistema afectó al color del zumo filtrado, que no se mantuvo constante durante el proceso, lo que supone una importante desventaja que es necesaria abordar. Como segunda aproximación, se evaluó el potencial de la inmovilización de una molécula bioactiva sobre membranas de celulosa, para mejorar la capacidad de retención microbiana del material celulósico, así como permitir su reutilización. Los filtros de celulosa funcionalizados con poliaminas demostraron ser eficaces en la eliminación de patógenos en agua, debido a las cargas positivas generadas por los grupos amina inmovilizados en la superficie de las membranas, que atraen y retienen las bacterias cargadas negativamente. Dada la fácil preparación y procedimiento de uso de las membranas de celulosa funcionalizadas con poliaminas, éstas podrían ser consideradas una buena opción para el desarrollo de sistemas de tratamiento de aguas in situ, rápidos, de fácil manejo y de bajo coste. El segundo capítulo describe el desarrollo y aplicación de partículas de sílice funcionalizadas con compuestos de aceites esenciales, con el fin de diseñar coadyuvantes de filtración con actividad antimicrobiana. La filtración de diversas matrices alimentarias (agua, cerveza y zumo de manzana) a través de los soportes funcionalizados con los antimicrobianos naturales demostró ser eficaz en la reducción del recuento de la cepa patógena Escherichia coli, así como frente a la microflora endógena de la cerveza y el zumo (bacterias acidolácticas, aerobios mesófilos, psicrófilos, mohos y levaduras). La eficacia en el control microbiano se debe a la combinación de la adsorción física y la inactivación por contacto con los compuestos de aceites esenciales inmovilizados. Además, la evaluación de las propiedades físico-químicas y sensoriales de los alimentos líquidos demostró un efecto poco significativo, éste depende del tamaño de las partículas de sílice usadas y de la molécula bioactiva inmovilizada. Por lo tanto, el sistema de conservaci� / [CA] En la present tesi doctoral s'ha avaluat l'ús de nous suports cel·lulòsics i silicis com a sistemes de filtració per a l'estabilització i conservació d'aliments líquids, amb la finalitat d'afrontar dos grans reptes de la indústria de begudes. D'una banda, evitar o minimitzar els canvis en les propietats nutricionals, estructurals i organolèptiques dels aliments, ocasionats per la pasteurització tèrmica tradicional, i oferir una alternativa al problema de la baixa viabilitat deguda als alts costos d'inversió/producció en aplicar noves tecnologies no tèrmiques. Per això, aquesta tesi doctoral es centra en el desenvolupament i avaluació d'una nova tecnologia no tèrmica de conservació d'aliments líquids basada en la filtració. S'han desenvolupat sistemes de filtració a partir de suports cel·lulòsics i silicis, sense funcionalitzar o funcionalitzats amb compostos antimicrobians. En el primer capítol es va avaluar l'ús de materials de cel·lulosa com a suports filtrants per al tractament d'aliments líquids. Com a primera aproximació es va desenvolupar un material porós nano-micro tubular a partir de l'extracció i deslignificació del material cel·lulòsic present en el cor o raquis de la panolla de dacsa. L'ús d'aquest suport va resultar ser efectiu com a material filtrant per al tractament d'aigua i suc de taronja, en un sistema de flux continu, eliminant la càrrega microbiana. L'aplicació d'aquest suport com a sistema de filtració presenta diversos avantatges com la seua capacitat de retenció microbiana, la reutilització de subproductes de la dacsa i, per tant, el seu respecte al medi ambient. No obstant això, seria necessari optimitzar el procés de filtrat per a evitar la freqüent obturació dels seus porus que va requerir diversos cicles de rentada durant el procés, així com establir un mètode de regeneració del material per a incrementar la seua vida útil. A més, aquest sistema va afectar el color del suc filtrat, que no es va mantenir constant durant el procés, la qual cosa suposa un important desavantatge que és necessari abordar. Com a segona aproximació, es va avaluar el potencial de la immobilització d'una molècula bioactiva sobre membranes de cel·lulosa, per a millorar la capacitat de retenció microbiana del material cel·lulòsic, així com permetre la seua reutilització. Els filtres de cel·lulosa funcionalitzats amb poliamines van demostrar ser eficaces en l'eliminació de patògens en aigua, a causa de les càrregues positives generades pels grups amina immobilitzats en la superfície de les membranes, que atrauen i retenen els bacteris carregats negativament. Donada la fàcil preparació i procediment d'ús de les membranes de cel·lulosa funcionalitzades amb poliamines, aquestes podrien ser considerades una bona opció per al desenvolupament de sistemes de tractament d'aigües in situ, ràpids, de fàcil maneig i de baix cost. El segon capítol descriu el desenvolupament i aplicació de partícules de sílice funcionalitzades amb compostos d'olis essencials, amb la finalitat de dissenyar coadjuvants de filtració amb activitat antimicrobiana. La filtració de diverses matrius alimentàries (aigua, cervesa i suc de poma) a través dels suports funcionalitzats amb els antimicrobians naturals va demostrar ser eficaç en la reducció del recompte del cep patogen Escherichia coli, així com enfront de la microflora endògena de la cervesa i el suc (bacteris àcid làctics, aerobis mesòfils, psicròfils, floridures i llevats). L'eficàcia en el control microbià es deu a la combinació de l'adsorció física i la inactivació per contacte amb els compostos d'olis essencials immobilitzats. A més, l'avaluació de les propietats fisicoquímiques i sensorials dels aliments líquids estudiats va demostrar un efecte poc significatiu, aquest depèn de la grandària de les partícules de sílice usades i de la molècula bioactiva immobilitzada. Per tant, el sistema de conserv / [EN] In the present doctoral thesis the use of new cellulosic and silica supports as filtering systems for the stabilization and preservation of liquid foods has been evaluated to overcome two major challenges of the beverage industry. On the one hand, avoid or minimize the changes in the nutritional, structural and organoleptic properties of food caused by traditional thermal pasteurization, and offer an alternative to the problem of low viability due to high investment/production costs when applying new non-thermal technologies. Therefore, this doctoral thesis focuses on the development and evaluation of a new non-thermal technology for the preservation of liquid foods based on filtration. The filtering systems have been developed from cellulosic and silica supports, non-modified or functionalized with antimicrobial compounds. In the first chapter, the use of cellulose materials as filtering supports for the treatment of liquid foods was evaluated. As first approximation, a porous nano-micro tubular material was developed from the extraction and delignification of the cellulosic material present in the corn stalk. The use of this support was effective as filtering material for the treatment of water and orange juice, in a continuous flow system, eliminating the microbial load. The application of this support as filtering system has several advantages, such as its microbial retention capacity, the reuse of corn by-products and, therefore, its respect for the environment. However, it would be necessary to optimize the filtering process to avoid the frequent clogging of its pores that required several washing cycles during the process, as well as to establish a method of material regeneration to increase its life. In addition, this system affected the color of the filtered juice, which did not remain constant during the process, representing an important disadvantage that must be addressed. As a second approach, the potential of the immobilization of a bioactive molecule on cellulose membranes was evaluated to improve the microbial retention capacity of the cellulosic material, as well as to allow its reuse. The cellulose filters functionalized with polyamines proved to be effective in eliminating pathogens in water, due to the positive charges generated by the amine groups immobilized on the surface of the membranes, which attract and retain the negatively charged bacteria. Given the easy preparation and usage of the polyamines-functionalized cellulose membranes, these could be considered a good option for the development of fast, easy to use and low cost in situ water treatment systems. The second chapter describes the development and application of silica particles functionalized with essential oil components to design filtering aids with antimicrobial activity. The filtration of various food matrices (water, beer and apple juice) through the supports functionalized with natural antimicrobials proved to be effective in reducing the load of the pathogenic strain Escherichia coli, as well as reducing the endogenous microflora of beer and the juice (lactic acid bacteria, mesophilic, psychrophilic, mold and yeast). The removal capability is due to the combination of physical adsorption and contact inactivation with the essential oil compounds immobilized. In addition, the evaluation of the physicochemical and sensory properties of the liquid foods studied showed a not significant effect, it depends on the size of the silica particles used and the immobilized bioactive molecule. Therefore, the proposed preservation system has a high potential for cold beverage pasteurization processes. / N. Peña-Gomez would like to thank for financial support in the frame of her PhD project to Operational Programme of the European Social Fund (ESF) 2014-2020, the Agencia Estatal de Investigación, Generalitat Valenciana and FEDER-EU (Projects RTI2018-101599-B-C21 and AGL2015-70235-C2-1-R). The authors also thank the Electronic Microscopy & Microanalysis Laboratory at Patras University for support. / Peña Gomez, N. (2020). Development of polymeric and silica filtering materials functionalized with antimicrobial compounds for the elimination of microorganisms in liquid food [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/137041 / TESIS / Compendio

The dissertation is focused on the study of intercellular interactions in skin tumors. It is based on 5 original publications that cover several topics. We studied the origin of tumor-associated fibroblasts concerning the primary tumor population. We demonstrated using a mouse model that tumor-associated fibroblasts are produced from the host organism and thus did not arise from transformation directly from tumor cells. We also investigated the relationship between tumor-associated fibroblasts and keratinocytes. We have shown that tumor-associated melanoma fibroblasts affect keratinocytes which, under their influence, acquire the features typically observed in migrating cells and cells undergoing epithelial-mesenchymal transition. We studied the interactions between healthy fibroblasts and tumor cells. We have demonstrated that fibroblasts acquired from healthy skin from a patient suffering from melanoma are significantly different from control fibroblasts of healthy donors in the expression profile. Changes in distal fibroblasts support the view of melanoma as a systemic disease. We have further demonstrated that melanoma-associated fibroblasts do not carry a BRAF mutation, in contrast to BRAF positivity of melanoma cells. And therefore, they did not arise from the transition from melanoma. The...

A Araucaria angustifolia é uma conífera nativa do Brasil. Em função da sua intensa exploração florestal, a espécie ocupa apenas 2% de sua vegetação natural. Neste sistema, a aplicação de técnicas biotecnológicas, como a embriogênese somática, podem ser integradas a programas de melhoramento genético e conservação. A similaridade entre a embriogênese somática e zigótica, tem sido utilizada para o estabelecimento de estudos visando o aperfeiçoamento do cultivo in vitro dos embriões somáticos, bem como para um maior conhecimento dos aspectos moleculares e fisiológicos que regulam a embriogênese. O metabolismo de poliaminas (PAs), mais especificamente putrescina, espermidina e espermina, tem se mostrado como fundamental para a compreensão e evolução da embriogênese zigótica e somática. Entretanto, a biossíntese das PAs e seu envolvimento nos vários processos biológicos que regulam a embriogênese, são pouco conhecidas em coníferas. Inserido nessa perspectiva, o presente trabalho teve como objetivo o estudo do metabolismo de PAs durante três estádios de desenvolvimento da semente (contendo as fases da embriogênese inicial até a tardia) e na proliferação de linhagens embriogênicas com diferentes potenciais embriogênicos de A. angustifolia. Foram investigados: a) os perfis de PAs (livres e conjugadas) e aminoácidos; b) determinação da via preferencial da biossíntese de putrescina, através da atividade enzimática da arginina descarboxilase (ADC) e ornitina descarboxilase (ODC); c) identificação e caracterização do padrão de expressão dos genes envolvidos no metabolismo de PAs; e d) a identificação das relações entre os perfis de PAs e aminoácidos presentes nas sementes das matrizes, e sua potencial influência nas fases de indução, proliferação e maturação dos embriões somáticos. Durante a embriogênese zigótica, a expressão dos genes AaADC (arginina descarboxilase) e AaSAMDC (S-adenosilmetionina descarboxilase) aumentaram no estádio cotiledonar, juntamente com o aumento de PAs. A biossíntese da putrescina é realizada preferencialmente via ADC, enquanto que a citrulina foi o principal aminoácido presente nas sementes. Em relação ao metabolismo de PAs nas culturas embriogênicas, os dados obtidos demonstraram que a arginina e ornitina parecem ter diferentes funções em cada linhagem testada. Na linhagem com alto potencial embriogênico, a arginina parece estar associada com a ativação dos genes relacionados ao catabolismo de PAs (AaPAO2, AaCuAO e AaALDH), enquanto que esse efeito não foi observado na linhagem bloqueada. A ODC tem uma maior atividade na linhagem responsiva, enquanto que na linhagem bloqueada, as atividades da ADC e ODC são similares. Dependendo da matriz foram observados diferentes perfis de PAs e aminoácidos, sendo estes perfis relacionados com as taxas de indução, proliferação e desenvolvimento dos embriões somáticos. Putrescina total, ornitina e asparagina foram os metabólitos diferencialmente identificados entre as matrizes, os quais podem ser propostos como marcadores bioquímicos para a seleção de matrizes com alto potencial para a embriogênese somática. Os resultados obtidos fornecem informações relevantes e inéditas sobre o metabolismo de PAs e aminoácidos na embriogênese zigótica e somática de A. angustifolia, bem como fornece novos subsídios para o aprimoramento das condições artificiais utilizadas para o desenvolvimento dos embriões somáticos / The Araucaria angustifolia is a native conifer species of Brazil. Due to its intense exploitation, the species cover only 2% of its original forest area. In this system, biotechnological tools, like somatic embryogenesis, may be integrated into breeding and conservation programs. The similarity between zygotic and somatic embryogenesis have been used to establishment of studies in order to optimization of somatic embryos in vitro culture, as well as for a better understanding of physiologic and molecular aspects that modulates the embryogenesis. The metabolism of polyamines (PAs), specifically putrescine, spermidine and spermine, has been demonstrated as fundamental for the comprehension and evolution of zygotic and somatic embryogenesis. However, the biosynthetic pathways of PAs and their involvement in various biological process that regulate the embryogenesis are little known in conifers. Inserted in this perspective, the aim of the current work was to study the metabolism of PAs during three seeds development stages (containing the early till late embryogenesis phases) and in proliferation of cell lines with different embryogenic potential of A. angustifolia. Were investigated: a) PAs (free and conjugated) and amino acids profiles; b) determination of preferential pathway for putrescine biosynthesis, through enzymatic activity of arginine decarboxylase (ADC) and ornithine decarboxylase (ODC); c) identification and characterization of gene expression profile of genes related to metabolism of PAs; and d) identification of the relationship between PAs and amino acids profiles in seeds of mother plants, and their potential influence in initiation, proliferation and maturation phases of somatic embryos. During the zygotic embryogenesis, AaADC (arginine decarboxylase) and AaSAMDC (S-adenosylmethionine decarboxylase) genes were up-regulated at cotyledonary stage along with the increasing of PAs. The biosynthesis of putrescine is performed preferentially by ADC pathway, while citrulline was the main amino acid recorded during the seed development. Regarding the metabolism of PAs in embryogenic cultures, the data demonstrated that arginine and ornithine seem to have different functions in each cell line tested. In cell line with high embryogenic potential, arginine seems to be associated to activation of genes related to PAs catabolism (AaPAO2, AaCuAO e AaALDH), while in blocked cell line this effect was not observed. ODC has a higher enzymatic activity in responsive cell line, while in blocked cell line, both ADC and ODC activities are similar. Depending of mother plant, were observed different PAs and amino acids profiles, being these profiles related with the rate of initiation, proliferation and maturation of somatic embryos. Total putrescine, ornithine and asparagine were the differentially metabolites identified between the mother plants, which can be proposed as biochemical marker to select mother plant with high potential to somatic embryogenesis. The results obtained provide relevant and inedited information about the metabolism of PAs and amino acids in zygotic and somatic embryogenesis of A. angustifolia, as well as provide news subsidies for optimization of in vitro conditions for somatic embryos development

Orientador: Giuseppina Pace Pereira Lima / Banca: João Domingos Rodrigues / Banca: Denise Laschi / Banca: Terezinha Rangel Câmara / Banca: José Luis Mosca / Abstract: Beehive ginger inflorescences have yellow bracts when they are young, which are ornamental and are specially used in gardening projects and as cut flowers. However, there are crop management and postharvest factors that affect the expansion of the species. So, the objective of this work was to study some physiological postharvest aspect of beehive ginger inflorescences grown in the lower middle São Francisco river basin. Flower stems just harvested were submitted to different treatments (distilled water; 75 mg L-1 of silver nitrate - AgNO3; 1000 mg L-1 of cobalt chloride - CoCl2; 5 mg L-1 de GA3 - Progibb® and 10 mg L-1 of 6-benzylamino purine - BAP), in an environment with controlled temperature and humidity, for 15 days. The longevity was monitored from non-destructive analysis (grading scale, fresh weight, consumption and pH of the preservative solution) as well as destructive ones (peroxidase and polyphenol oxidase activity, total soluble and reducing carbohydrates, phenols, putrescine, spermine and spermidine content). The non-destructive analysis showed that the beehive ginger stems treated with gibberelin and silver nitrate presented a better visual aspect according to the grading scale, the ones treated with AgNO3 absorbed a greater volume of the solution during the experimental period, while the ones treated with silver nitrate and BAP had a greater fresh weight. The smallest variation of the preservative solution pH took place with the treatments containing the plant regulators. The destructive analysis revealed that the inflorescences maintained in preservative solutions with gibberelin and distilled water kept their stocks of total soluble sugars for 3 days longer than the stems submitted to the other treatments. The contents of reducing sugars increased 7 considerably in inflorescences treated with cytokinin. The BAP promoted alterations in the activity of the... (Complete abstract click electronic access below) / Doutor

La phytoremédiation constitue une nouvelle technologie permettant de dépolluer les sols contaminés par l'utilisation de plantes. Parmi les différents aspects possibles de cette méthode, figure la phytoextraction basée sur l'absorption et l'accumulation du polluant dans les parties aériennes. Pour être efficace, il est nécessaire de disposer de plantes présentant une biomasse élevée. L'objectif de ce travail visait à sélectionner des variétés de moutarde indienne (Brassica juncea L.) tolérantes et accumulatrices de cadmium.<br />La première partie du travail a consisté à mettre au point et à optimiser une méthode de régénération in vitro de plantes de moutarde indienne, à partir de couches cellulaires minces transversales (CCMTs) (influence de l'organe utilisé, de AgNO3 et de la benzylaminopurine). La régénération a été réalisée en appliquant une pression de sélection par le cadmium pour modifier la tolérance au métal. <br />La deuxième partie aborde l'effet des traitements par le cadmium : in vitro et en serre, sur le développement des plants. Une analyse des perturbations physiologiques et biochimiques observées a permis d'évaluer la tolérance des plants vis à vis du cadmium et indiquent que les plantes néoformées en présence de cadmium mettent en place un système d'exclusion du métal.<br />Dans la troisième partie, pour compléter l'étude précédente sur des plantes néoformées, l'effet du cadmium a été testé sur des plantes de B. juncea directement issues de semis. Plusieurs paramètres physiologiques et biochimiques, caractéristiques des stress, ont été étudiés (activité gaïacol peroxydase, peroxydation des lipides, pigments, acides aminés libres, proline glucides, polyamines libres et conjuguées). En raison de leurs propriétés anti-oxydantes, une attention particulière a été portée aux polyamines dont l'application exogène permet d'envisager son utilisation pour améliorer la capacité d'accumulation du cadmium par les plantes.

Las poliaminas regulan procesos de crecimiento y diferenciación celular, y su desregulación está relacionada con diferentes patologías incluyendo el cáncer. Las antizimas (AZs) de ornitina descarboxilasa (ODC) inhiben tanto su biosíntesis, como su captación, regulando los niveles intracelulares de poliaminas. En esta tesis se ha caracterizado una nueva proteína inhibidora de antizimas (AZIN2) que posee alta homología con ODC y el inhibidor de antizimas previamente conocido (AZIN1). Esta nueva proteína está desprovista de actividad enzimática, pero es capaz de revertir la acción que las tres antizimas conocidas ejercen sobre la actividad ODC y la captación de poliaminas. A diferencia de sus proteínas homólogas, AZIN2 se localiza subcelularmente en el ERGIC, y se expresa específicamente en cerebro y testículo, pero de forma muy abundante en espermátidas y espermatozoides, al igual que AZ3, indicando que estas dos proteínas juegan un importante papel regulando los niveles de poliaminas durante la espermiogénesis. / Polyamines regulate cell growth and differentiation, and the alteration of their homeostasis is related to different diseases, including cancer. Ornithine decarboxylase (ODC) antizymes (AZs) regulate polyamine levels by inhibiting both their biosynthesis and the cellular uptake. In this work, a new ODC paralogue has been characterized as a novel antizyme inhibitor protein that has been named AZIN2. This protein lacks decarboxylating activity, but it is able to reverse the action of any of the three antizymes on ODC activity and polyamine uptake. Unlike its homologue proteins ODC and AZIN1, AZIN2 is located in the ERGIC, and it is specifically expressed in brain and testes. The abundant expression in spermatids and spermatozoa, concomitantly with AZ3, suggests that both proteins may play an important role in regulating polyamine levels during spermiogenesis

The polyamines spermine and spermidine play important roles in many cellular processes, and unusual levels of these polyamines have been associated with numerous human diseases. Spermidine/spermine N¹-acetyltransferase (SSAT) is an enzyme involved in polyamine regulation, where acetylation of polyamines by SSAT ultimately leads to their degradation or export from the cell. In this dissertation, x-ray crystallography and nuclear magnetic resonance (NMR) are used to provide insights into the structure and function of this important enzyme. X-ray crystallography provided two distinct views of SSAT: one of the enzyme in complex with coenzyme A (CoA), and another of the enzyme in complex with CoA and the polyamine spermine. Together, the two structures reveal structural plasticity in the active site of the enzyme. The complex with spermine provides a direct view of polyamine binding by SSAT, and shows that the enzyme relies heavily on associated water molecules to bind spermine; these water molecules also appear to form a "proton relay" between the primary amine of spermine and the side-chain of a conserved glutamate residue. Guided by the structural results, NMR methods were used to test hypotheses regarding the enzyme mechanism of SSAT. The activity of the enzyme over a range of solution conditions, and towards different polyamine substrates, was determined; the effects of mutating single amino acids in the enzyme were also evaluated. The enzyme appeared to be most active between pH 8.5 and 9.5, and mutation of the aforementioned glutamate significantly altered this behavior. This suggests the glutamate is directly involved in the acetyltransfer reaction, where it likely functions as a catalytic base though the proton relay in the enzyme active site. These studies advance our general understanding of how polyamines are regulated in mammalian cells, and have the potential to assist in developing new therapeutic options for human diseases involving polyamines. / text