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京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20219号 / 医博第4178号 / 新制||医||1019(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 小西 靖彦, 教授 小池 薫, 教授 坂井 義治 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13449号 / 論医博第2242号 / 新制||医||1054(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 川口 義弥, 教授 妹尾 浩, 教授 柳田 素子 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Polyamines are a class of essential nutrients involved in many basic cellular processes such as gene expression, cell proliferation, and apoptosis. Without polyamines, cell growth is delayed or halted. Cancerous cells require an abundance of polyamines through a combination of synthesis and transport from the extracellular environment. An FDA-approved drug, D,L-α-difluoromethylornithine (DFMO), blocks polyamine synthesis but is ineffective at inhibiting cell growth due to polyamine transport. Thus, there is a need to develop drugs that inhibit polyamine transport to use in combination with DFMO. Surprisingly, little is known about the polyamine transport system in humans and other eukaryotes. Understanding the transport system would allow us to identify compounds that inhibit polyamine transport, which could then be used in tandem with DFMO to treat cancer. Our laboratory has identified one gene in Drosophila, called CG32000, as a component of this transport system, and numerous other candidate genes remain to be tested. To better characterize this system, this project investigated the ability of the Drosophila transport system to take up a toxic polyamine analogue called norspermidine, with the initial goal of developing a new screening method to find polyamine transport genes. My experiments have demonstrated significant differences in norspermidine uptake and toxicity between C. elegans and Drosophila which may imply a secondary polyamine transport system in higher eukaryotes. In the long term, it is hoped that this thesis will facilitate the development of more effective cancer medications by providing new information about the polyamine transport system.

La synthèse et la vectorisation d’agents anticancéreux constituent des axes de recherche majeurs du LCSN. De nombreux composés naturels possèdent des propriétés anticancéreuses, mais ils sont abandonnés en raison de leur manque de sélectivité vis-à-vis des cellules cancéreuses ou de leur faible biodisponibilité. Ainsi, un grand intérêt est actuellement porté sur le développement de médicaments spécifiquement vectorisés vers les cellules cancéreuses. Les vecteurs utilisés dans ce travail sont des dérivés de polyamines et des nano objets de type β-cyclodextrines / nanocristaux de cellulose (β-CD/CNCx). Les polyamines vont permettre un ciblage actif des cellules cancéreuses grâce au système de transport de polyamine (PTS) surexprimé dans ces cellules. Les nano objets vont cibler spécifiquement les tumeurs via un ciblage passif dû à l’effet EPR. Les principes actifs employés dans cette étude sont des flavonoïdes, et plus particulièrement des chalcones. En effet, les flavonoïdes, qui constituent une large famille de composés phénoliques naturels, sont connus pour leurs nombreux effets biologiques comme les activités antioxydantes, anti-inflammatoires et anti-prolifératives.L’intérêt du LCSN à la fois pour les chalcones et les agents anticancéreux nous a conduits à concevoir de nouveaux composés antiprolifératifs vectorisés. Ce travail présente dans un premier temps la synthèse de chalcones et l’obtention de dérivés couplés aux différents vecteurs décrits précédemment (motifs polyaminés,β-CD/CNCx) ; un travail sur la synthèse d’une bis-chalcone via le couplage de Suzuki est également exposé.L’ensemble des molécules obtenues est caractérisé par des analyses RMN 1H, 13C et HRMS. Dans une seconde partie, nous présentons l’ensemble des évaluations biologiques des composés précédemment obtenus. Ces évaluations sont réalisées par un test de viabilité cellulaire (test MTT) sur quatre lignées cancéreuses : deux colorectales (HT-29 et HCT-116) et deux prostatiques (PC-3 et DU-145). / Synthesis and vectorization of anticancer agents are major research themes of LCSN. Many natural compoundspossess anti-cancer properties, but they are dropped because of their lack of selectivity to cancer cells or theirlow bioavailability. Thus, great interest is currently focused on the development of drugs specifically vectorizedto cancer cells. The vectors used in this work are polyamine derivatives and nano-objects type β-cyclodextrin /cellulose nanocrystals (β-CD/CNCx). Polyamines allow active targeting of cancer cells through the polyaminetransport system (PTS) overexpressed in these cells. Nano-objects specifically target tumors using a passivetargeting due to the EPR effect. Drugs used in this study are flavonoids, especially chalcones. Indeed,flavonoids, which constitute a large family of natural phenolic compounds, are known for their numerousbiological effects such as antioxidant, anti-inflammatory and anti-proliferative activities. The interest of LCSNfor both chalcones and anticancer agents led us to design new vectorized anti-proliferative compounds. Firstly,this work shows the synthesis of chalcones and their derivatives coupled to various above-described vectors(polyamines units, β-CD/CNCx); a work on the synthesis of a bis-chalcone through the Suzuki coupling reactionis also exposed. All molecules obtained are characterized by 1H NMR, 13C NMR and HRMS analysis. In thesecond part of this work, we present all biological evaluations of compounds previously obtained. Theseassessments are performed through a cell viability test (MTT test) on four cancer cell lines: two colorectal (HT-29 and HCT-116) and two prostate (PC-3 and DU-145) cell lines.

CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior / Polyamines and ethylene have been cited as important regulators of plant growth and development, and may be involved in plant defense against several abiotic stresses, such as salinity. To withstand with salt harmful effects, plants respond through a coordinated set of physiological and molecular responses to improve their performance under salinity. In order to test the hypothesis that salt tolerance degree in maize genotypes is related to changes in polyamine metabolism associated with ethylene production, two experiments were performed. In the first one, BR5033 (salt-tolerant) and BR5011 (salt-sensitive) maize genotypes were subjected to 80 mM NaCl stress to identify the pattern of ethylene production in leaves and roots. Two peaks of ethylene production at 5.5 h (phase I) and 12.5 h (phase II) after onset the salinity treatment were registered in salt-sensitive leaves; whereas only the first peak of ethylene synthesis was detected in salt-tolerant leaves. Surprisingly, the biphasic ethylene production in roots was much less pronounced than in leaves. In the second experiment, we sought to investigate whether the phases I and II of ethylene production alter the polyamine metabolism in the leaves of maize genotypes. In salt-tolerant genotype, the phase I of ethylene synthesis was associated with signaling events, as evidenced by increased H2O2 levels, which were generated by putrescine (Put) catabolism. An early signaling (at 5.5 h) in the salt-tolerant genotype seemed to be effective to suppress the second peak of ethylene production, known as âstress ethyleneâ. Yet, in the salt-sensitive genotype, the decreased H2O2 concentration during the phase I was associated with a marked increase in ethylene production, which was resulted from upregulation of acid 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) activity and ZmACO5 gene expression. At the phase I, the total polyamine content was increased by salinity in the salt-tolerant, whereas it was decreased in the salt-sensitive one. In the salt-tolerant genotype, the increased total polyamine was sustained by high spermine (Spm) and spermidine (Spd) contents, while the decay in the salt-sensitive genotype was due to the reductions of Put and Spd forms. Otherwise, in the phase II, no significant changes in the total polyamines in salt-tolerant genotype (it was likely due to conversion of Put to Spm/Spd), and decreases in salt-sensitive genotype were registered. Under stress conditions, the salinity-induced improvement of Spd and Spm (free and soluble conjugated forms) in salt-tolerant genotype was bigger than in salt-sensitive one, thus suggesting a key role of polyamines in the maize salt stress acclimation processes. Finally, we investigated if the lack of ethylene production during phase II in salt-tolerant genotype was correlated to improved antioxidant capacity. Salt stress dramatically increased the superoxide levels, the electrolyte leakage and lipid peroxidation, it being more pronounced in both leaves and roots of salt-sensitive genotype. On the other hand, under salinity, salt-tolerant genotype displayed a better performance of enzymatic and non-enzymatic antioxidant system, evidenced by a higher ascorbate and glutathione content and upregulation of superoxide dismutase, ascorbate peroxidase and guaiacol peroxidase activity. In conclusion, our results suggest that the ethylene is intimately involved in salt stress acclimation through activation of intricate signaling pathways mediated by H2O2 that is originated from polyamine catabolism. An efficient signal network raises the polyamine content and antioxidant capacity and is responsible, at least in part, for greater tolerance to salinity of BR5033 maize genotype. / Poliaminas e etileno sÃo reguladores do crescimento e desenvolvimento vegetal, que tambÃm estÃo envolvidos nas respostas de defesa das plantas contra estresses abiÃticos, dentre eles a salinidade. Para lidar com o estresse salino, as plantas realizam ajustes fisiolÃgicos, bioquÃmicos e moleculares, que podem resultar em sua aclimataÃÃo diante dessa condiÃÃo adversa, tornando o indivÃduo mais tolerante ao estresse, em comparaÃÃo Ãqueles que nÃo se encontram aclimatados. Essa pesquisa foi desenvolvida para testar à hipÃtese de que o grau de tolerÃncia à salinidade entre genÃtipos de milho envolve alteraÃÃes no metabolismo das poliaminas associadas à produÃÃo de etileno. Para isso, foram realizados dois experimentos. No primeiro, plantas de milho dos genÃtipos BR5011 (sensÃvel) e BR5033 (tolerante) foram submetidas ao estresse salino (NaCl a 80 mM) para identificar o padrÃo de produÃÃo de etileno em folhas e raÃzes. Nas folhas do genÃtipo sensÃvel, a salinidade intensificou a produÃÃo de etileno apÃs 5,5 h (fase I) e 12,5 h (fase II) apÃs o inÃcio do estresse, enquanto no tolerante isso aconteceu somente com 5,5 h. Nas raÃzes, embora tenha sido observada a produÃÃo bifÃsica de etileno no genÃtipo sensÃvel, esse processo foi muito menos intenso que nas folhas. O segundo experimento teve como objetivo principal investigar se a produÃÃo de etileno pela salinidade nas fases I e II resultava em alteraÃÃes no metabolismo das poliaminas nas folhas dos genÃtipos de milho. No genÃtipo tolerante, a fase I de produÃÃo de etileno foi associada à eventos de sinalizaÃÃo, dado o aumento dos teores de H2O2, mediado pelo catabolismo da putrescina (Put). Essa sinalizaÃÃo pareceu ser eficiente para suprimir a produÃÃo do etileno em condiÃÃes de estresse (fase II ou âetileno do estresseâ) nesse genÃtipo. Jà no sensÃvel, a diminuiÃÃo dos teores de H2O2 na fase I foi acompanhada por um aumento acentuado na produÃÃo do etileno, decorrente de acrÃscimos na atividade da enzima oxidase do Ãcido 1-carboxÃlico-1-aminociclopropano (ACO) e na expressÃo de transcritos do gene ZmACO5 (principal membro expresso). Em geral, a salinidade aumentou os teores de poliaminas totais no genÃtipo tolerante, enquanto reduziu no sensÃvel. Na fase I, na condiÃÃo salina quando comparada com o controle, os teores de poliaminas totais foram aumentados no genÃtipo tolerante enquanto no sensÃvel esses teores foram reduzidos. No genÃtipo tolerante, o aumento nos teores de poliaminas totais foi sustentado principalmente pelo aumento nos teores de espermina (Spm) e espermidina (Spd), enquanto a diminuiÃÃo observada no genÃtipo sensÃvel foi devida, sobretudo, Ãs reduÃÃes nas formas de Put e Spd. Jà na fase II, no genÃtipo tolerante nÃo houve alteraÃÃes nos teores totais de poliaminas (provavelmente, devido a utilizaÃÃo de Put para a sÃntese de Spm e Spd), enquanto no sensÃvel esses teores foram reduzidos. Sob condiÃÃes de salinidade, o aumento nas formas livre e conjugada solÃvel de Spm e Spd foi mais pronunciado no genÃtipo tolerante do que no sensÃvel, sugerindo assim importante papel para essas duas poliaminas nos processos de aclimataÃÃo ao estresse salino em plantas de milho. Por fim, foi investigado se a ausÃncia de produÃÃo do etileno na fase II, causado pela salinidade no genÃtipo tolerante, foi relacionada com uma melhor capacidade antioxidante. O estresse salino aumentou drasticamente os teores do radical superÃxido, o vazamento de eletrÃlitos e a peroxidaÃÃo lipÃdica, sendo isso mais pronunciado nas folhas e raÃzes do genÃtipo sensÃvel. De modo geral, o genÃtipo tolerante teve melhor desempenho do sistema antioxidante enzimÃtico e nÃo enzimÃtico, sob condiÃÃes de estresse salino, evidenciado pelos maiores incrementos nos teores de ascorbato e glutationa e na atividade das enzimas dismutase do superÃxido, peroxidase do ascorbato e peroxidase do guaiacol. Em conclusÃo, os resultados aqui apresentados sugerem que o etileno està intimamente envolvido na aclimataÃÃo ao estresse salino, por meio da ativaÃÃo de vias de sinalizaÃÃo mediadas pelo H2O2 produzido a partir do catabolismo de poliaminas. AlÃm disso, sugere-se que essa sinalizaÃÃo induz o aumento nos teores de poliaminas e melhor capacidade antioxidante no genÃtipo BR5033, sendo isto, pelo menos em parte, responsÃvel por sua maior tolerÃncia ao estresse salino, quando comparado ao BR5011.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Polyamines, such as spermidine and spermine, have been reported to improve memory retention through the activation of N-methyl-D-aspartate receptors (NMDAr). However whether polyamine agonists and antagonists alter extinction remains unclear. In the current study, we investigated whether spermidine and polyamine antagonists that selectively block the NR2B subunit at the NMDAr alter the extinction of contextual conditioned fear in male Wistar rats. While the bilateral intrahippocampal administration of exogenous spermidine (2 nmol/site) facilitated the extinction of fear conditioning, the injection of the antagonists arcaine (0.2 nmol/site), ifenprodil (20 nmol/site) and traxoprodil (0.2 nmol/site), disrupted fear extinction. NMDAr antagonists, at doses that had no effect per se, reversed the facilitatory effect of spermidine on fear extinction. These results suggest that exogenous and endogenous polyamines facilitate the extinction of contextual conditioned fear through activation of NR2B subunit-containing NMDAr in the hippocampus. Since extinction-based exposure therapy is widely used as treatment for a number of anxiety-related disorders, including phobias and post-traumatic stress, the currently reported facilitation of extinction by polyaminergic agents suggest these compounds as putative candidates for drug development. / As poliaminas, como espermidina e espermina, são aminas alifáticas que estão presentes no sistema nervoso central e que se ligam na subunidade NR2B do receptor N-metil-D-aspartato (rNMDA). Tem-se demonstrado que a administração sistêmica, intrahipocampal e intraamígdala de poliaminas melhoram a aquisição e retenção da memória em ratos. Entretanto, seu efeito sobre a extinção do medo condicionado não foi investigado. No presente estudo, investigamos se a administração intrahipocampal de espermidina e de antagonistas seletivos para a subunidade NR2B do rNMDA alteram a extinção do medo condicionado contextual em ratos Wistar machos. A administração intrahipocampal de espermidina (2 nmol/sítio) facilitou a extinção do medo condicionado, enquanto que a injeção dos antagonistas do rNMDA, arcaína (0,2 nmol/sítio), ifenprodil (20 nmol/sítio) e traxoprodil (0,2 nmol/sítio), bloquearam a extinção do medo condicionado contextual. Já a administração dos antagonistas do rNMDA, em doses sem efeito per se, reverteu a facilitação da extinção induzida por espermidina. Estes resultados sugerem que as poliaminas facilitam a extinção do medo condicionado contextual através da ativação da subunidade NR2B do rNMDA hipocampal. Tendo em vista que a terapia baseada em exposição é um método amplamente utilizado como tratamento para diversos tipos de distúrbios relacionados com ansiedade, incluindo fobias e estresse pós-traumático, a facilitação da extinção causada pela administração de espermidina coloca este composto com um possível candidato para o desenvolvimento de novos fármacos para o tratamento destas patologias.

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.

Snyder-Robinson syndrome (SRS) is an X-linked neurodegenerative disorder affecting males. The disease appears early in childhood with symptoms like bone abnormalities, reduced muscle mass, and mobility issues. It results from disrupted polyamine biosynthesis, which is due to a mutation in spermine synthase (Sms). This causes an abnormally elevated Spermidine: Spermine ratios. There is no approved SRS treatment, rather only management of symptoms. A genetic mouse model for the SmsG56S mutation found in some SRS patients was characterized, and a novel spermine Prodrug treatment was tested. The hypothesis is that male SmsG56S mutant mice exhibit polyamine dysregulation and SRS traits, while Prodrug intervention may rebalance abnormal ratios and facilitate a normalized phenotype. Mice were phenotyped in pure C57BL/6J, mixed C3H, and backcrossed C57BL/6J x C3H backgrounds. Lethality of the mutation, especially in C57BL/6J mice, was an obstacle. Viable mutant mice exhibited reduced body weight, typically smaller size, and lower bone densities compared to age-matched wild-type mice. Prodrug treatment was performed using different dosing strategies and in all backgrounds. Upon histological examination, testes and bone exhibited subtle defects in affected mutant male mice, while no detectable differences were found in skeletal muscle, liver, or kidney compared to wild-type mice. Acute prodrug treatment using oral gavage 5 times per week over 2 weeks was found to rebalance the Spermidine: Spermine ratio. Repeated efforts to dose Prodrug over a longer 6-8 week duration in mice required lower intraperitoneal doses but outcomes may have been moderated by the mice background. Overall, the SmsG56S phenotype in the testes and bone are consistent with other mutant Sms models, although C57BL/6J mice seemed to be more sensitive to the SmsG56S mutation. Further testing of Prodrug is needed, including in younger mice and for a longer duration of treatment, to evaluate Prodrug effectiveness in improving traits in SmsG56S mice.

Nas abordagens biotecnológicas de propagação de plantas, os meios de cultura devem ter uma composição química adequada à essa finalidade permitindo a otimização da produção. Como a bananeira (Musa sp.) é exigente em potássio, a busca do nível adequado desse macronutriente envolve não somente o comprometimento com o nível dos outros nutrientes (balanço iônico), mas também a relação entre eles. Para acompanhar os efeitos fisiológicos das relações de vários teores de K com os outros macro e micronutrientes é que explantes caulinares dos cvs. Nanica e Prata Anã foram cultivados em meio MS modificado em presença de BAP, sacarose, vitaminas, agar, suplementado com 6 diferentes doses de K: 5, 10, 15, 20, 25 e 30 mM, sendo que a dose 20 mM corresponde à concentração de K existente no MS básico, que foi adotado como controle.Foram feitas análises de massa de matéria seca (MMS),macro e micronutrientes na parte aérea, raiz e plântulas inteiras. Ao final do experimento foi determinado o número de plântulas e calculado o valor das relações N/K, K/P, K/Ca, K/Mg, K/Ca+Mg, K/S, K/Cu, K/Fe, K/Mn, K/Zn.Foram também dosados os teores da diamina putrescina e de poliaminas, e calculada a relação K/putrescina. Todos os parâmetros foram analisados segundo um delineamento experimental inteiramente casualizado. As plântulas que se desenvolveram em baixas concentrações de K apresentaram sintomas visuais de deficiência, como clorose e necrose das folhas mais velhas. Os cultivares apresentaram diferenças quantitativas entre si tanto em relação aos valores de MMS como em número de plântulas, relacionados às doses de K presentes nos meios de cultura. Em ambos os cultivares foi observada uma relação direta entre o desenvolvimento das plântulas e as concentrações de K com otimização ao redor de K 15 a 20 mM. Os teores de putrescina e de poliaminas foram maiores nos níveis mais baixos de K, atingindo o máximo na dose de K 5 mM. Em K 20 mM ocorreram maiores valores de MMS e em K 15 mM maior número de plântulas regeneradas. O íon K em geral foi mais intensamente absorvido do que os outros macro e micronutrientes sendo que estes tiveram sua absorção diminuída devido provavelmente a um efeito de diluição de seus teores pelo crescimento das plântulas in vitro. Esses resultados, inclusive os obtidos nas demais relações entre K e os outros macro e micronutrientes, as quais sempre foram crescentes (de K 5 a 30 mM), corroboram a essencialidade desse nutriente para os cvs. Nanica e Prata Anã. / Potassium is required in high dosis by the banana plant (Musa sp.) and interacts with other macro and micronutrients present in the medium in which banana tissues are maintained in vitro condition,with consequent modifications in the plant cell metabolism, mainly in nitrogen compounds, such as proteins and amino acids. When K is present in concentrations lower than the required, diamines such as putrescine, and polyamines, such as spermidine and spermine are formed. In order to establish the best dosis of K and follow the physiological consequences of the relationships N/K, K/P, K/Ca, K/Mg, K/Ca+Mg, K/S, K/Cu, K/Fe, K/Mn, K/Zn and K/putrescine, shoot apex of two banana cvs. Nanica and Prata Anã were maintained in asseptic conditions in modified MS media in the presence of 6 different dosis of K: 5, 10, 15, 20, 25 and 30 mM, K 20 mM being the K concentration in basic MS medium, and then transferred to rooting media with the same different K dosis. Dry wt., macro and micronutrients were measured in the shoots, roots and the intire plantlet, and number of plantlets produced determined, the data being analysed estatistically. Putrescine and polyamines were also determined. Visual symptoms of K deficiency such as clorosis and necrosis in the older leaves of all plantlets under low dosis of K were observed. The levels of putrescine and polyamines increased as K decreased in the medium, reaching the maximum value at K 5 mM, both cultivars presenting similar bahavior in relation to the diamine in some K concentrations. Quantitative differences were obtained among the two cultivars pertained to dry wt. values, number of in vitro regenerated banana plantlets and K concentration with optimization around K 15 and 20 mM. In general K absorption was more intense than the other nutrients, the absorption of the later being decreased probably due to a dilution effect of their values as the banana plantlets developed in vitro. These results, including those pertained to the relationships between K and the other nutrients, which always were high (from K 5 to 30 mM), corroborate the importance of potassium ion to the banana cvs. Nanica and Prata Anã.