Conselho Nacional de Desenvolvimento Científico e Tecnológico / Polyamines (putrescine, spermidine and spermine) are aliphatic amines produced by the action of ornithine descarboxylase (ODC), a rate-limiting and protein kinease C (PKC)-regulated step in polyamine synthesis. Since the levels of polyamines were found to be high in synovial fluid of arthritic patients, the aim of the present study was to identify the role of peripherally produced polyamines in the model of inflammatory pain induced by adjuvant arthritis. The subcutaneous injection of complete Freund s adjuvant (CFA, 50 μL/paw) caused the development of mechanical allodynia and edema. Moreover, it increased ODC expression and activity as well as PKC activation. The previous administration of the selective ODC inhibitor DFMO (10 μmol/paw) was capable of preventing the development of allodynia and edema as well as the increase in ODC activity produced by CFA injection. Furthermore, the previous administration of the PKC inhibitor GF109203X (1 nmol/paw) reduced allodynia and the increase of ODC activity in animals injected with CFA. Accordingly with the synthesis inhibition, we have observed that subcutaneous injection of putrescine (10 μmol/paw), spermidine (3-10 μmol/paw) or spermine (0.3-3 μmol/paw) into the rat paw also caused mechanical allodynia and edema. The present results suggest that endogenously synthesized polyamines are involved in the development of nociception and edema caused by the adjuvant. Moreover, the polyamine production in inflammatory site seems to be related with the increase in ODC activity stimulated by PKC activation. Thus, the control of polyamine synthesis and action could be an interesting target to control inflammatory pain. / Poliaminas (putrescina, espermidina e espermina) são aminas alifáticas produzidas pela ação da ornitina descarboxilase (ODC), enzima limitante e proteína quinase C (PKC), passo regulatório da síntese de poliaminas. Desde que níveis elevados de poliaminas foram encontrados no fluído sinovial em pacientes com artrite, o objetivo do presente estudo foi investigar a produção de poliaminas perifericamente em modelo de dor inflamatória induzido por CFA. A injeção subcutânea do adjuvante completo de Freund (CFA, 50 μL/pata) causou o desenvolvimento de alodínia mecânica e edema, bem como um aumento na expressão e atividade da ODC e na ativação da PKC. A administração prévia do inibidor seletivo da ODC, o DMFO (10 μmol/pata), foi capaz de prevenir o desenvolvimento da alodínia e edema, bem como o aumento da atividade produzida pela injeção de CFA. Além disso, a pré-administração do inibidor da PKC o GF109203 (1 nmol/pata), reduziu a alodínia e o aumento da atividade da ODC em animais injetados com CFA. De acordo com a inibição da síntese, também observamos que a injeção subcutânea de putrescina (10 μmol/pata), espermidina (3-10 μmol/pata) ou espermina (0,3-3 μmol/pata) na pata de ratos desenvolveu alodínia mecânica e edema. O presente estudo sugere que as poliaminas sintetizadas endogenamente estão envolvidas no desenvolvimento da nocicepção e edema causado pelo CFA. Além disso, as poliaminas produzidas nos sítios de inflamação estão relacionadas com o aumento na atividade da ODC estimulada pela ativação da PKC. Assim, o controle da síntese das poliaminas e função poderia ser um interessante alvo para o controle da dor inflamatória.

Eukaryotic cells tightly regulate metabolism in order to sustain normal processes. Dysregulation of cellular metabolism is associated with multiple diseases including cancer. Polyamine metabolism is a tightly regulated process that is co-opted by multiple cancers for selective growth advantages. Polyamines are small organic molecules with two or more amino groups attached, whose biosynthesis is initiated by ornithine decarboxylase (ODC). Although much is known regarding the effects of polyamine metabolism and ODC on cellular processes, little is known regarding the intracellular signaling events that are regulated by polyamines. Clinical studies demonstrated that the ODC inhibitor difluromethylornithine (DFMO) was an effective chemopreventative strategy causing a reduction of colon adenomas in patients with prior colon polyps. However, the molecular mechanisms leading to this reduction are unknown. This dissertation provides mechanistic insight into the biological roles of the polyamines and show that these amines are regulators of multiple non-coding RNAs involved in cellular responses including effects on the let-7 microRNA family. Moreover, the polyamine modified translation factor eIF5A is demonstrated to regulate the oncofetal factor LIN28. This work also indicates that polyamines regulate the mTOR pathway and suggests alternative signaling nodes for polyamine-mediated regulation of cellular processes. Overall, these findings support the notion that polyamines are oncometabolites that are targetable and serve as a promising approach to manipulate oncogenic signaling for cancer therapy.

Abstract Fetal alcohol syndrome (FAS) is a pattern of anomalies in affected children due to maternal alcohol administration at vulnerable stages of fetal development. Intrauterine growth restriction and facial malformation are the presenting phenotypes of FAS. In this investigation, novel pathomechanisms of intrauterine growth restriction and facial malformation were the primary aims. We found by a FAS mouse model that AceCS1 gene expression and polyamines are the immediate targets of fetal alcohol exposure. The AceCS1 product is a precursor for lipid synthesis and protein acetylation and possibly, for polycation acetylation. We cloned the Mus musculus nuclear-cytosolic AceCS1 gene, and showed that its expression is developmentally regulated with a dynamic localization in the cytosolic and nuclear compartment. The enzyme plays an essential role in de novo synthesis of acetyl Coenzyme A. Fetal alcohol administration targets nutrient supplying networks, which are localized at critical barriers. The main findings were reduced surface of the labyrinthine zone, destruction of gap junctions in the hemotrichorial placenta, reduced syncytiotrophoblastic cell layers and loosening of interaction between cell layers and embryo endothelial cells, reduced Reichert’s membrane thickness with discontinued Reichert’s trophoblast and loss of interaction by Reichert’s-parietal cells, reduction of capillary network and reduced vascularization in the brain area, and perturbed neural crest migration and formation of neural tube defect. Alteration of angiogenesis -regulating proteins such as VEGF, PlGF, PECAM was detected in FAS, with no significant changes in placental angiogenesis of the labyrinthine zone, but up-regulation of VEGF/PlGF caused permeability changes in the placenta and yolk sac. On the other hand, the PECAM pool in embryos’ brain was reduced, which in turn led to decreased angiogenesis and vascularization. / Tiivistelmä Sikiön alkoholisyndrooma (engl. Fetal alcohol syndrome, FAS) on joukko muutoksia, joita esiintyy äidin raskaudenaikaisen alkoholin käytön seurauksena, kun käyttö osuu sikiökehityksen kannalta kriittiseen vaiheeseen. Kohdunsisäisen kasvun rajoittuminen ja kasvojen epämuodostumat ovat FAS:n tyypillisimpiä ilmentymiä. Tässä tutkimuksessa pyrittiin löytämään uusia patomekanismeja kohdunsisäisen kasvun rajoittumiselle ja kasvojen epämuodostumille. Hiiren FAS-mallin avulla selvisi, että sikiön altistuminen alkoholille vaikuttaa suoraan AceCS1-geenin ilmentymiseen ja polyamiinien pitoisuuteen. AceCS1-geenin tuote on esiaste lipidien synteesissä ja proteiinien asetylaatiossa sekä mahdollisesti myös polykationien asetylaatiossa. Työssä myös kloonattiin hiiren (Mus musculus) AceCS1-geeni, jonka tuotetta esiintyy sekä tumassa että solulimassa. Lisäksi osoitettiin, että geenin ekspressio oli kehityksen aikana säädelty tuottamaan entsyymiä dynaamisesti eri paikkoihin solussa. Entsyymillä on lisäksi merkittävä osuus asetyyli-koentsyymi-A:n de novo–synteesissä. Sikiön altistuminen alkoholille kohdistuu sellaisten ravintoaineiden saatavuuteen, jotka sijaitsevat kriittisesti tärkeissä kudosrajapinnoissa. Päälöydöksinä olivat vähentynyt labyrinttikudoksen pinta-ala, gap-liitosten tuhoutuminen istukan veriesteessä (hemotrichorial?), ohentunut trofoblastisolujen kerros ja Reichertin kalvon paksuus, harventunut hiusverisuonten verkosto sekä verisuonitus aivojen alueella sekä hermopienan solujen siirtymishäiriö ja hermostoputken sulkeutumishäiriö. Verisuonten muodostumista (angiogeneesiä) säätelevien proteiinien (kuten VEGF, PlGF, PECAM) muutoksia todettiin FAS:ssa, mutta merkittäviä muutoksia ei havaittu istukan verisuonten muodostumisessa. VEGF/PlGF-suhteen suureneminen muutti istukan ja ruskuaispussin verisuonten läpäisevyyttä. Toisaalta sikiöiden aivojen PECAM-määrä pieneni, mikä johti verisuonten ja verisuoniverkoston muodostumisen vähenemiseen.

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.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / In Alzheimer s disease (AD), β-amyloid peptide (Aβ) has been linked with synaptic loss and cognitive dysfunction, albeit the precise mechanism remains unknown. An involvement of N-Methyl-D-Aspartate receptors (NMDAR) in AD is proposed, since its inhibition attenuates some aspects of AD s neuropathology. In this regard, polyamines, like spermidine and spermine, positive modulators of NMDARs, have been shown to have both concentration and synthesis increased by Aβ. Using the novel object recognition task we showed that negative modulation of polyamine system, been trough blockade of its binding site at NMDAR by arcaine (0.02 nmol/site), traxoprodil (0.002 nmol/site), or inhibition of polyamine synthesis by DFMO (2.7 nmol/site), reverses Aβ25-35-induced memory impairment in mice. The activation of polyamine binding site at NMDAR located at extrasynaptic sites might underlie the cognitive deficits of Aβ25-35-treated mice, since incubation of hippocampal neuron cultures with spermidine (400 μM) or Aβ25-35 (10 μM) significantly increased nuclear accumulation of jacob protein, a marker of extrasynaptic NMDAR activation. Moreover, traxoprodil (4nM), arcaine (4 μM) or DFMO (5 μM) blocked the Aβ-induced jacob nuclear translocation. Activation of extrasynaptic NMDAR in neurons leads to striping of synaptic contacts and simplification of neuronal cytoarchitecture. Incubation of hippocampal neuron cultures with traxoprodil (4 Nm), arcaine (4 μM) or DFMO (5 μM) reversed the deleterious effects of Aβ25-35 on dendritic spine number and spine morphology. We also evaluated the involvement of A-type K+ currents on the Aβ25-35-induced memory impairment. Administration of Tx3-1 (3 100 pmol/site), a selective IA blocker, restored memory of mice injected with Aβ25-35 and tested on the novel object recognition task The reversal of memory impairment and the protective effect on dendritic spine alterations exerted by the modulators of the polyamine system suggest the polyamine binding site at extrasynaptic NMDAR a potential player in Aβ-induced cognitive deficit. / O peptídeo β-amiloide (Aβ), reconhecido como agente tóxico na Doença de Alzheimer (DA) é implicado como causador de danos cognitivos e sinápticos, apesar de os mecanismos não serem completamente compreendidos. O envolvimento do receptor N-metil-D-aspartato (NMDA) na DA é sugerido, visto que o seu bloqueio atenua alguns aspectos neuropatológicos da DA. Nesse contexto, tem sido demonstrado que as poliaminas, como espermidina e espermina, moduladores positivos do receptor NMDA, possuem níveis e síntese elevada tanto no cérebro de pacientes com DA como em preparações in vitro utilizando o peptídeo Aβ. Neste estudo demonstrou-se que a modulação do sistema das poliaminas, através do bloqueio do seu sítio de ligação no receptor NMDA por arcaína (0,02 nmol/sítio), traxoprodil (0,002 nmol/sítio) ou da inibição de sua síntese por DFMO (2,7 nmol/sítio), reverte o déficit cognitivo induzido pela injeção de Aβ25-35 em camundongos testados na tarefa de reconhecimento de objetos. A ativação do sítio de ligação das poliaminas em receptores NMDA extrassinápticos pode subjazer o déficit cognitivo de camundongos injetados com Aβ25-35, visto que a incubação de culturas primárias de neurônios hipocampais com espermidina (400 μM), NMDA (200 μM) ou Aβ25-35 (10 μM) aumenta o acúmulo nuclear de jacob, um marcador de ativação de receptores NMDA extrassinápticos, de maneira significante. Ademais, traxoprodil (4 nM), arcaína (4 μM) ou DFMO (5 μM) bloquearam o acúmulo nuclear de jacob induzido por Aβ. A ativação de receptores NMDA extrassinápticos em neurônios leva a simplificação da citoarquitetura neuronal e a diminuição de contatos sinápticos. Aqui demonstrou-se que a incubação de culturas de neurônios hipocampais com traxoprodil (4 nM), arcaína (4 μM) ou DFMO (5 μM) reverte as alterações na a densidade e morfologia de espinhas dendríticas induzido pela incubação com Aβ25-35. Ainda, também avaliou-se o envolvimento de correntes de K+ do tipo A no déficit cognitivo induzido pela injeção i.c.v. de Aβ25-35. A administração de Tx3-1 (3 100 pmol/sítio), um bloqueador seletivo de correntes IA, reverteu o prejuízo de memória de camundongos injetados com Aβ25-35 e testados na tarefa de reconhecimento de objetos. A reversão dos danos cognitivos e sinápticos induzidos por Aβ25-35 através da modulação do sistema das poliaminas sugere a estimulação do sítio de ligação das poliaminas no receptor NMDA, possivelmente extrassínaptico, como um dos mecanimos por trás do déficit cognitivo induzido pelo peptídeo Aβ.