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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.

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This dissertation is a compilation of published works and a manuscript that seek to understand the possible role of polyamines in the regulation of capsule in Streptococcus pneumoniae (Spn, pneumococcus). Spn remains a major health risk worldwide while the capsule is widely recognized as the principal virulence factor. Polyamines on the other hand are small hydrocarbon molecules known to regulate a number of cellular processes in bacteria. This work investigates the impact of deletion of polyamine biosynthesis gene, SP_0916 (cadA, lysine decarboxylase at the time of first and second publication), on protein expression and the capsule biosynthesis of virulent pneumococcal serotype 4 (TIGR4). We identify loss of capsular polysaccharide (CPS) in the deletion strain and based on proteomics results, we hypothesized that a shift in metabolism that favors the pentose phosphate pathway (PPP) over glycolytic pathway, that could reduce the availability of precursors for CPS had occurred. Comparison of transcriptomic and untargeted metabolomics profile of ∆SP_0916 with TIGR4 shows impaired glycolysis and Leloir pathways that provide CPS precursors, in the mutant strain. Furthermore, gene expression changes indicate possible reduction of common polyamines (cadaverine, putrescine, spermidine and spermine). Targeted metabolomics analysis confirmed reduced levels of polyamines in SP_0916. However, the result suggests that SP_0916 encodes an arginine decarboxylase, contrary to its existing annotation as a lysine decarboxylase in many bioinformatics databases. Biochemical characterization of the purified protein encoded by SP_0916 confirms that it is indeed catalyzes arginine decarboxylation, and exogenous supplementation of agmatine, the product of the reaction, successfully restores capsule biosynthesis. This study fixes an error in annotation of the TIGR4 genome and further establishes the essentiality of agmatine, a product of arginine decarboxylation as the key polyamine molecule modulating pneumococcal capsule. We later compared the impact of deletion of polyamine synthesis by gene deletion (ΔSP_0916) with chemical inhibition of synthesis using α- difluoromethylornithine (DFMO), in multiple pneumococcal serotypes. Results of this dissertation confirmed that pneumococcal pathways impacted by the disruption of polyamine biosynthesis either by gene deletion or chemical intervention are conserved and could regulate capsule synthesis.

Polyamines are polycationic molecules that perform many functions essential to cell growth and proliferation. These compounds can be synthesized inside the cell or taken into the cell exogenously. Many tumor types have been shown to contain elevated polyamine levels and an activated polyamine transporter (PAT) for importing exogenous polyamines. Thus, the PAT represents a potential target for anti-cancer strategies. To date, the mechanism of polyamine uptake into cells of multicellular eukaryotes is poorly understood, primarily because the only PAT proteins that have been identified are in the unicellular organisms Escherichia coli, Saccharomyces cerevisiae, and the protozoan parasite Leishmania major. In order to identify a PAT, in a higher eukaryote, a novel Drosophila imaginal disc epithelial development assay was employed. In this assay, development of the imaginal disc epithelium is inhibited by polyamine-toxin conjugates with results (PAT selectivity and toxicity) similar to those shown in vertebrate Chinese Hamster Ovary (CHO) cells. Using the Gene Ontogeny database at Fly Base, 39 candidates for a Drosophila PAT were identified based on their putative function as cation transport proteins or polyamine transport proteins. Twenty of the candidate genes were selected for further analysis based on their expression in imaginal discs and availability of strains carrying mutations in these genes. Imaginal discs dissected from the candidate mutant strains were tested for defects in polyamine uptake following exposure to polyamine-toxin conjugates. Mutations in two genes, Orct2 and CG9413, conferred increased resistance to the polyamine-toxin in the epithelial assay, as would be expected for imaginal disc cells defective in polyamine transport. Therefore, these two genes represent candidates for the polyamine transporter in a multicellular eukaryote

L’émergence de comportements collectifs cellulaires n’est pas bien comprise. Nous l’abordons dans deux systèmes biologiques. A l'échelle du micromètre lors de la constriction de l’anneau cytokinétique, nous montrons que des complexes d’acto-myosine s’auto-organisent sous forme d’agrégats dans la levure à fission et dans la cellule de mammifères. Ces auto-organisations découlent de règles d'interactions communes mais pour des fonctions distinctes, le transport et la génération de stress respectivement. A l'échelle de 100 micromètres, nous observons des pulsations corrélées de cellules épithéliales. Nous montrons les rôles du frottement avec la surface, et le couplage entre l’aire cellulaire, sa hauteur et sa contractilité. Nous présentons aussi deux études, des polyamines synthétiques pour étudier la polymérisation d'actine in vivo, puis l’inversion de sens dans la migration - la ratchetaxie. Cette thèse illustre l'importance des phénomènes physiques dans la dynamique cellulaire. / The emergence of collective behavior from the interaction of individual units is not clear. In this thesis, we address this question in two different systems at different scales. At the micrometer scale during cytokinetic ring constriction, we show that acto-myosin self-organizes into rotating and static clusters in fission yeast and mammalian cells. These self-organizations arise from common interaction rules, but to serve distinct functions, transport and stress generation respectively. At 100 micrometers scale, we report correlated pulsations of cells in an epithelial monolayer. We show the key roles of substrate friction, and the tight coupling between cell area, cell height and contractility. We also present two other studies: synthetic polyamines for studying actin polymerization in vivo, and direction reversal in single cell migration during ratchetaxis. Altogether, this PhD illustrates the importance of physical phenomena in cellular dynamics.

Les muscles permettent de générer force et mouvements et ont des fonctions métaboliques importantes. Mon travail a consisté à caractériser le rôle et les mécanismes d’actions des androgènes dans le muscle strié. Nous avons montré que l’ablation du récepteur des androgènes dans les myofibres n’affecte pas la masse musculaire car à la fois les voies anaboliques (IGF1) et cataboliques (myostatine) sont dérégulées. Cependant, l’absence du récepteur dans les myofibres diminue l’hypertrophie musculaire induite par une surcharge mécanique et limite l’atrophie induite par les glucocorticoïdes. Son ablation augmente également l’autophagie, entrainant une déstructuration des sarcomères, conduisant à une diminution de la force musculaire. De plus, sa délétion diminue la vitesse d’absorption du glucose lors d’une surcharge glucidique. Le récepteur des androgènes dans les myofibres régule donc la masse et la force musculaire, ainsi que l’import du glucose. / Muscles generate strength and movement, and have important metabolic functions. The aim of my work was to characterize the role and mechanisms of action of androgen receptor in skeletal muscle. We show that ablation of the androgen receptor in skeletal muscle myofibers does not affect muscle mass as both anabolic (IGF1) and catabolic pathways (myostatin) are deregulated. However, the absence of this receptor in myofibers decreases muscle hypertrophy induced by mechanical overload and limits glucocorticoids-induced muscle atrophy. Its ablation also increases autophagy, leading to sacromeres destructuration, resulting in decreased muscle strength. Moreover, its deletion reduced the rate of glucose absorption during a glucidic overload. Thus, myofibres androgen receptor regulates muscle mass and strength, as well as glucose import.

Muitos estudos têm sido direcionados para gerar melhorias na cultura de cana-de-açúcar, sendo uma das alternativas o uso de técnicas biotecnológicas, como a embriogênese somática (ES). O objetivo deste projeto foi estudar a multiplicação celular na ES de cana-de-açúcar da variedade SP80-3280. Culturas embriogênicas (CE) e não-embriogênicas (CNE) foram monitoradas e parâmetros bioquímicos foram analisados. Foi observado que CE e CNE apresentaram crescimento máximo aos 24 dias de cultivo, sendo que as CE apresentaram maior incremento em matéria fresca. Dos carboidratos analisados, a sacarose foi predominante em CE, enquanto a glicose predominou em CNE. Os conteúdos de glicose e frutose variaram simultaneamente em CE. As concentrações de amido, poliaminas (Pas) totais e a razão entre PAs [Razão PA= Put/(Spd+Spm)] apresentaram maiores valores nas CNE, sendo a espermidina a poliamina predominante em CE e putrescina em CNE. O conteúdo de proteínas totais foi significativamente maior em CE, em todas as fases de crescimento. / Many studies have been directed to generate improvements in the sugarcane cultures, one of the alternative use of biotechnology techniques such as somatic embryogenesis (SE). The objective of this project was to study the cell multiplication in SE of sugarcane, variety SP80-3280. Embryogenic cultures (EC) and non-embryogenic (NEC) were monitored and biochemical parameters were analyzed. Was observed that EC and NEC showed maximum growth to 24 days of culture, and the EC showed greater increase in fresh weight. Of the carbohydrates tested, sucrose was predominant in EC, while glucose was predominant in the NEC. The contents of glucose and fructose varied simultaneously in EC. The concentrations of starch, polyamines (PAs) and the ratio of total PAs [Ratio PA = Put / (Spd + Spm)] showed higher values in the NEC, and the spermidine is predominant polyamine to EC and putrescine is predominant in NEC. The content of total protein was significantly higher in CE, at all stages of growth.

Embora o cultivo de tomate seja muito antigo e amplamente distribuído, ainda enfrenta desafios para o melhoramento dos níveis de produção e, da qualidade para o processamento e consumo fresco. A grande maioria das características de interesse agronômico está determinada por loci de caracteres quantitativos (QTL), dificultando ainda mais a identificação e transferência gênica. Diversas características tornam o tomateiro um bom modelo para estudos de dissecação dos determinantes genéticos de QTL. Primeiro, a disponibilidade de fontes de germoplasma selvagens ainda inexploradas que podem aumentar a variabilidade genética, somada à possibilidade de cruzamento entre espécies não simpátricas e à autogamia. Segundo, a grande quantidade de informação genética como mapas, coleções de ESTs, QTL, e populações de mapeamento. Terceiro, o genoma de S. lycopersicum está completamente sequenciado. Por fim, devido a suas diferenças morfogenéticas em relação à espécie modelo Arabidopsis thaliana, o tomate se torna uma alternativa para estudos de eudicotiledonias, principalmente em estudos relacionadas a metabolismo de frutos carnosos. A partir da abundante plataforma de dados disponível e por meio de ferramentas de genômica, e genética reversa, este trabalho aborda o estudo de duas regiões do genoma do tomateiro envolvidas no metabolismo de aminoácidos e na determinação da estrutura da parede celular. O estudo de genômica comparativa de uma região do cromossomo 7 permitiu revelar a perfeita sintenia existente entre S. lycopersicum e a espécies selvagem S. pennellii e estimar o tempo de divergência em 2,7 MAA. Complementarmente, foi possível determinar que as diferenças fenotípicas entre as espécies são maiormente devidas a mudanças nas regiões regulatórias e à presença de SNPs. O estudo funcional do gene LFP (Low Free Putrescine) permitiu caracterizar uma proteína plastidial, até o momento desconhecida em tomateiro, que participa do metabolismo de poliaminas. O silenciamento de LFP resultou na redução da disponibilidade de putrescina livre e no aumento da biomassa vegetativa. Por outro lado, os resultados obtidos do estudo do gene GAUT4 (galacturonosiltransferase 4) demonstraram que a enzima codificada se localiza em Golgi e participa do metabolismo das pectinas. Em frutos, a redução dos níveis de GAUT4 resultou na diminuição de pectina e na alteração da sua composição, porém estes se apresentaram mais firmes. Adicionalmente, o silenciamento do gene GAUT4 modificou o particionamento de açúcares provocando o aumento de massa vegetativa em detrimento do índice de colheita, revelando assim um mecanismo regulatório que comunica o metabolismo da parede celular ao controle da relação fonte-dreno. Desta forma, os resultados obtidos aportam dados fundamentais para a melhor compreensão de caracteres de interesse agronômico, assim como de processos fisiológicos complexos e pouco explorados até o momento no tomateiro / Although tomato is an old and widely distributed culture, it still faces challenges to improve production levels and quality for processing and consumption. The vast majority of agronomical important characteristics are determine by quantitative trait loci (QTL), further hindering the gene identification and transfer. Several features make tomato a good model for studying the genetic determinants underneath QTL. First, the availability of unexploited sources of wild germplasm that can increase genetic variability, coupled with the possibility of interbreeding between no sympatric species and autogamy. Second, the large amount of available genetic information as maps, EST collections, QTL, and an extensive collection of mapping populations. Third, the genome of S. lycopersicum is completely sequenced. Finally, due to their morphogenetic differences in relation to model species Arabidopsis thaliana, tomato becomes an alternative to eudicotyledons studies, especially in studies related to fleshy fruits metabolism. From the abundant data platform available and by means of genomic tools, and reverse genetics, this work addresses the study of two tomato genomic regions involved in amino acids and cell wall metabolisms. The comparative genomic study in a region of chromosome 7 has revealed the perfect synteny between S. lycopersicum and the wild species S. pennellii, and estimated the time of divergence between both species in 2.7 MYA. Additionally, it was possible to determine that the phenotypic differences between species are mostly due to changes in regulatory regions and the presence of SNPs. The functional study of LFP gene (Low Free Putrescine) allowed us to characterize a plastid protein, yet unknown in tomato, which participates in the metabolism of polyamines. The LFP silencing resulted in reduced availability of free putrescine and increased vegetative biomass. Furthermore, the functional characterization of the GAUT4 (galacturonosyltransferase 4) gene demonstrated that the encoded enzyme is located in the Golgi apparatus and participates in the pectin metabolism. In fruits, the reduced levels of GAUT4 resulted in decreased pectin and in the change of its composition. Additionally, GAUT4 gene silencing modified sugars partitioning leading to an increased vegetative biomass together with a drastic reduction of the harvest index. Thus, revealing a regulatory mechanism that communicates the cell wall metabolism to source-sink relationship control. Concluding, the results obtained contribute to a better understanding of agronomical important traits, as well as of complex physiological processes little explored in tomato so far

Infectious diseases such as malaria and relapsing fever borreliosis (RF), cause severe human mortality and morbidity in developing countries. Malaria, caused by Plasmodium spp. parasites, is estimated by the World Health Organization to cause 1.5-2.7 million deaths annually. RF, caused by Borrelia spirochetes, has the highest prevalence described for any bacterial disease in Africa, with infection outcomes ranging from asymptomatic to fatal. RF borreliosis manifests in humans as a recurring fever and with other symptoms very similar to those of malaria. RF borreliosis has been regarded as a transient infection of the blood. However, B. duttonii exploits the brain as an immunoprivileged site escaping the host immune response while spirochetes in the blood are cleared. To investigate whether residual bacteria are dormant or actively dividing, mice with residual brain infection were administered ceftriaxone, a β-lactam antibiotic interfering with cell wall synthesis. Hence, it only affects actively dividing bacteria. Ceftriaxone eradicated brain RF infection in all treated mice, demonstrating that the bacteria are actively multiplying rather than in a dormant state. The findings support the therapeutic use of ceftriaxone for RF neuroborreliosis since penetration into cerebrospinal fluid is greater for ceftriaxone than for the often recommended doxycycline. The clinical features of malaria and RF are similar and diagnosis is further complicated by the frequently occurring concomitant malaria-RF infections. Therefore, we established a mouse model to study the pathogenesis and immunological response to Plasmodium/Borrelia mixed infection. Interestingly, malaria was suppressed in the co-infected animals whereas spirochete numbers were elevated 21-fold. The immune response in the concomitantly infected mice was polarized towards malaria leaving the spirochetes unharmed. Mice with co-infections also exhibited severe anemia and internal damages, probably attributed to escalating spirochete numbers. A secondary malaria infection reactivated the residual brain RF infection in 60% of the mice. This highlights the importance of co-infections as diagnostic pitfalls as well as the need for novel treatment strategies. Currently there is no commercial malaria vaccine and increasing drug resistance presents an urgent need for new malaria chemotherapeutics. Blood-stage malaria parasites are rapidly growing with high metabolic and biosynthetic activity, making them highly sensitive to limitations in polyamine supply. Disrupting polyamine synthesis in vivo with trans-4-methylcyclohexylamine (4MCHA) eradicated the malaria infection gradually, resulting in protective immunity. This leads the way for further biochemical and pharmacological development of the polyamine inhibitor 4MCHA and similar compounds as antimalarial drugs