Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The reactivation of a memory results in its destabilization, requiring a process of memory reconsolidation to maintain it. Spermidine is an endogenous aliphatic amine with polycationic structure that modulates N-methyl-D-aspartate (NMDA) receptor activity and improves memory. Recent evidence suggests that systemic administration of spermidine improves the reconsolidation of fear memory. Here we determined whether the calcium-dependent protein kinase (PKC) signaling pathway is involved in the improvement of fear memory reconsolidation induced by intrahippocampal (ih) administration of spermidine in rats. Male Wistar rats were trained in a fear conditioning apparatus using a 0.4 mA footshock as unconditioned stimulus. Twenty-four hours after training, animals were re-exposed to the apparatus in the absence of shock (reactivation session). Immediately after the reactivation session, spermidine (2-200 ρmol/site); the PKC inhibitor, 3-[1- (dimethylaminopropyl)indol-3-yl]-4-(indol-3-yl) maleimide hydrochloride (GF 109203X, 0.3 - 30 ρg/site); the antagonist of the polyamine-binding site at the NMDA receptor, arcaine (0.2 - 200 ρmol/site) or the PKC activator, phorbol 12-myristate 13-acetate (PMA, 0.02 - 2 nmol/site) were injected intra-hipocampally (i.h.). Testing was carried out in the same apparatus, twenty-four hours after reactivation. Freezing scores at testing were considered a measure of memory. While the post-reactivation administration of spermidine (20 and 200 ρmol/site) improved, GF 109203X (1, 10 and 30 ρg/site) impaired memory reconsolidation. GF 109203X (0.3 ρg/site) prevented spermidine (200 ρmol/site)-induced improvement of memory reconsolidation. The post-reactivation administration of arcaine (200 pmol/site) impaired and PMA (2 nmol/site) improved memory reconsolidation. PMA (0.2 nmol/site) prevented arcaine (200 ρmol/site)-induced impairment of memory reconsolidation. The protein synthesis inhibitor anisomycin (2 μg/site) prevented spermidine (200 ρmol/site)-induced improvement of memory reconsolidation. These drugs had no effect on memory if they were administered in the absence of reactivation. These results suggest that the enhancement of memory reconsolidation induced by the administration of spermidine involves PKC activation. / A reativação de uma memória resulta na sua desestabilização, que exigem um processo de reconsolidação de memória para mantê-la. A espermidina é uma amina alifática endógena com estrutura policatiônica que modula a atividade do receptor Nmetil- D-aspartato (NMDA) e melhora a memória. Evidências recentes sugerem que a administração sistêmica de espermidina melhora a reconsolidação da memória de medo. No entanto não se sabe se a administração intra hipocampal de espermidina melhora a reconsolidação da memória e nem se a proteína cinase dependente de cálcio (PKC) e síntese proteica estão envolvidas neste efeito. Portanto, no presente estudo verificou-se o envolvimento da PKC e da síntese proteica na melhora da reconsolidação da memória do medo induzida pela administração intrahipocampal (ih) de espermidina em ratos. Ratos Wistar machos foram treinados na tarefa de medo condicionado contextual utilizando-se choque de 0,4 mA como estímulo incondicionado. Vinte e quatro horas após o treino, os animais foram re-expostos ao aparelho na ausência de choque (sessão reativação). Imediatamente após a sessão de reativação foram administradas, por via ih, espermidina (2-200 ρmol/sítio); o inibidor da PKC, 3- [1- (dimetilaminopropil) indol-3-il] -4- (indol-3-il) maleimida (GF 109203X, 0,3-30 ρg/sítio); o antagonista do sítio de ligação das poliaminas no receptor de NMDA, arcaína (0,2-200 ρmol/sítio) ou o ativador de PKC, 12-miristato 13-acetato de forbol (PMA, 0,02-2 nmol/sítio). Os testes foram realizados no mesmo aparelho, 24 horas após a sessão de reativação. O tempo de imobilidade dos animais durante o teste foi considerado como medida de memória. Enquanto a administração de espermidina (20 e 200 ρmol/sítio) melhorou, GF 109203X (1, 10 e 30 ρg/sítio) prejudicou a reconsolidação da memória. O GF 109203X (0,3 ρg/sítio) preveniu a melhora da reconsolidação da memória induzida por espermidina (200 ρmol/sítio). A administração da arcaína (200 pmol/sítio) prejudicou e PMA (2 nmol/sítio) melhorou a reconsolidação da memória. O PMA (0,2 nmol/sítio) preveniu o prejuízo na reconsolidação da memória induzido por arcaína (200 ρmol/sítio). O inibidor de síntese de proteica, anisomicina (2 ug/sítio) preveniu a melhora da reconsolidação da memória induzida por espermidina (200 ρmol/sítio). Estas drogas não tiveram nenhum efeito sobre a memória quando foram administrados na ausência da sessão de reativação. Estes resultados sugerem que a melhora da reconsolidação da memória induzida pela administração ih de espermidina envolve a síntese proteica e a ativação de PKC.

Spermine (SPM) is an aliphatic amine which contains four nucleophilic centers and is found in all eukaryotic cells, including nervous cells. It belongs to the group of polyamines, which are molecules associated with both neuroprotection and neurotoxicity. The aim of this study was to investigate the effects of spermine on some parameters of toxicity induced by striatal administration of quinolinic acid (QA), an experimental model of Huntington s disease in adult and male Wistar rats. The intrastriatal administration of QA (180 nmol/site) induced contralateral rotations and increase the number of contralateral body swings. The previous striatal administration of SPM caused mixed effects: at the dose of 0.1 nmol/site increased the number of contralateral rotations; but at 10 nmol/site it reduced both the number of rotations and the contralateral body swings induced by QA. The mechanism by which SPM decreases these motor alterations is probably through its interaction with the NMDA receptor, since the co-administration with arcaine (antagonist of polyamine binding sites on this receptor) reversed its protective effect. The increase of protein carbonyl content induced by QA (180 nmol/site) in striatum of rats was prevented by the administration of 10 nmol/site of SPM. Besides, the bilateral striatal injection of QA (180 nmol/site) impaired the performance in the recognition memory task. The post-training striatal administration of SPM (0.1 and 1 nmol/site) reversed the QA-induced cognitive deficit. It was also evaluated whether spermine prevented QA-induced neuromorphological alterations. QA caused striatal neuronal degeneration and reactive astrogliosis. SPM, at the dose that improved the cognitive performance (0.1 nmol/site), had no effect on striatal neuronal degeneration but reversed the intense astrocytic reaction induced by QA. These results suggest that SPM has neuroprotective properties, presenting a dose dependent pattern of polyamine, in this experimental model of Huntington disease. / A espermina (SPM) é uma amina alifática, contendo quatro centros nucleofílicos e é encontrada em todas as células eucarióticas, incluindo células nervosas. Ela pertence ao grupo das poliaminas, moléculas responsáveis tanto por efeitos neuroprotetores quanto neurotóxicos. O objetivo do presente trabalho foi investigar os efeitos da SPM sobre alguns parâmetros de toxicidade induzidos pela administração estriatal de ácido quinolínico (AQ), um modelo experimental da doença de Huntington em ratos Wistar machos adultos. A administração intraestriatal unilateral de AQ (180 nmol/sítio) induziu o aparecimento de rotações contralaterais e aumento do percentual de balanços corporais contralaterais. A prévia administração estriatal de SPM mostrou efeitos diversos: na dose de 0,1 nmol/sítio aumentou o número de rotações; porém na dose de 10 nmol/sítio ela diminuiu tanto o número de rotações quanto o percentual de balanços corporais contralaterais induzidos pelo AQ. O mecanismo pelo qual a SPM diminui estas alterações motoras é, provavelmente, devido à sua interação com o receptor NMDA, uma vez que sua co-administração com a arcaína (antagonista do sítio das poliaminas neste receptor) reverteu o efeito protetor da mesma. A administração de 10 nmol/sítio de SPM preveniu o aumento do conteúdo de proteína carbonil induzida pela injeção de AQ (180 nmol/sítio) no estriado de ratos. Além disso, foi observado prejuízo cognitivo na tarefa de reconhecimento de objetos após a injeção estriatal bilateral de AQ (180 nmol/sítio). A administração estriatal póstreino de SPM (0,1 e 1 nmol/sítio) reverteu este déficit cognitivo induzido pelo AQ. Para avaliação das alterações neuromorfológicas neste modelo foram observadas degeneração neuronal e reação astrocitária. O AQ aumentou significativamente a degeneração de neurônios estriatais e a astrogliose reativa. A SPM, na menor dose que melhorou o desempenho cognitivo (0,1 nmol/sítio), não teve efeito sobre a degeneração neuronal estriatal; no entanto, ela foi capaz de reverter a intensa reação astrocitária induzida pela injeção de AQ. Estes resultados sugerem que a SPM tem propriedades neuroprotetoras, que apresentam um padrão dependente da dose da poliamina, neste modelo experimental da doença de Huntington.

[pt] Este trabalho descreve a síntese e caracterização de compostos de Cu(II), Co(II) e Cd(II). As técnicas de caracterização utilizadas foram análise elementar, termogravimetria, UV-vis, espectroscopia de infravermelho, RMN (para complexos de Cd(II)), EPR (para complexos de cobre (II)) e difração de raio X, quando obtidos cristais. A primeira parte do trabalho foi a síntese de um novo complexo: [Cu(PCr)(H2O)], e os resultados sugerem que a PCr age como um ligante tridentado (o átomo de nitrogênio do grupo guanidino e os átomos de oxigênio dos grupos fosfato e carboxilato como sendo os átomos doadores). A quarta posição na esfera de coordenação é ocupada por uma molécula de água. Estes resultados foram confirmados através de cálculos computacionais (DFT/B3LYP:6- 311G procedimento teórico) e mostram que o cobre(II) está tetracoordenado com uma geometria quadrática plana. A segunda parte deste trabalho foi a síntese de quatro novos complexos do tipo [Cu(ATP)(poliamina)], contendo como ligante as poliaminas (PA): etilenodiamina, 1,3- diaminopropano, espermidina, espermina e o ATP. Os valores dos parâmetros paralelos de EPR para os complexos mostram que o íon cobre(II) está complexado através dos oxigênios dos fosfatos do ATP. Os dados da TG indicam que nos omplexos estão presentes moléculas de água de hidratação. A parte final deste trabalho foi o estudo das interações dos sistemas entre as poliaminas e os tetraclorometalatos. Quatorze novos compostos de Cu(II), Co(II) e Cd(II) de estequiometria [MCl4(poliamina)] contendo, além das poliaminas já citadas, a poliamina putrescina foram preparados. Um complexo de Co(II) com a estequiometria [CoCl2(H2O)4]Put.2HCl, foi preparado e formou cristais. / [en] This work describes the synthesis and characterization of Cu (II), Co(II) e Cd(II) compounds. The characterization was performed by means of elemental and thermogravimetric analysis, UV-vis, IR, RMN (for Cd(II) compounds), EPR (for Cu(II) compounds) and X-ray diffractometry (for crystals). The first part of this work was the synthesis of one new complex : [Cu(PCr)(H2O)] and the results suggest that PCr is acting as a tridentate ligand (the nitrogen atom of the guanidine group and the oxygen atom of the phosphate group and the carboxylate group being the donor atoms). The fourth position is occupied by a water molecule. These results were confirmed through computational calculations (DFT/B3LYP:6-311G theoretical procedure) and show that Cu(II) is tetracoordinated and arranged in a tetrahedric geometry. The second part of the study was the synthesis and characterization of four new complexes of the type [Cu(ATP)(polyamine)] containing as ligands the polyamines (PA): ethylenediamine, 1,3-diaminepropane, spermidine or spermine and ATP. The EPR parallel parameters values for the complexes show that Cu(II) is complexed through the oxygen atoms from the phosphates groups of ATP. TG data indicate that each complex has the presence of one water molecule of hydration. The final part of this work was the study of the interactions occurring in systems between tetrachlorometalates and the polyamines. Fourteen new compounds of Cu(II), Co(II) and Cd(II) of stoichiometry [MCl4(polyamine)] were prepared, containing all the polyamines cited before plus putrescine were prepared. One complex of Co(II) with the stoichiometry [CoCl2(H2O)4] Put.2HCl, has formed single crystals.

Abstract Changes in climate will impose abiotic stress on plant species, and eventually affect their distributional range. This is a particular challenge especially to tree species with long generation times and slow rates of evolution. In the boreal zone, most of these species also hold high economic value. The aim of the study was to enhance understanding of the abiotic stress coping strategies of Scots pine (Pinus sylvestris L.) in its vulnerable early growth phase. In particular, the study evaluates the role of polyamine (PA) metabolism of Scots pine under abiotic stress. Polyamines (PAs) are small, ubiquitous nitrogenous compounds involved in fundamental biological processes of plants such as growth, organogenesis, embryogenesis and abiotic and biotic stress defence. The effects of different water availabilities and spring frost temperatures on shoots and roots of young seedlings were investigated in two growth chamber experiments. Proembryogenic liquid cell culture system was established and used as a controllable platform. Effects of polyethylene glycol (PEG) induced osmotic stress was investigated at the cellular level in liquid cultures. Responses to treatments were evaluated by quantifying changes in the expression of PA metabolism, stress, cell death and cell division -related genes, metabolite concentrations, as well as stress-induced physiology and morphology. The results revealed that PA metabolism is tissue specific and strictly regulated. Drought stress induced the accumulation of putrescine (Put) in photosynthesizing tissues, whereas the prime response to spring frost was a reorganization of root growth. Generally, drought and osmotic stress decreased the expression of PA catabolizing genes, leading to consistent PA levels in tissues even under severe stress. To conclude, PAs have a protective role in maintaining the growth and development of pine tissues subjected to abiotic stress. / Tiivistelmä Ilmastonmuutos vaikuttaa eri kasvilajien levinneisyyteen. Nopeasti muuttuva ilmasto on haaste erityisesti metsäpuulajeille, jotka pitkän sukupolvenvälin takia kehittyvät evolutiivisesti hitaasti. Erilaisten ympäristötekijöiden aiheuttamien puolustusaineenvaihdunnan muutosten tunteminen metsäpuilla on tulevaisuuden kannalta myös taloudellisesti tärkeää. Työn tavoitteena oli selvittää millainen merkitys polyamiineilla on metsämännyn (Pinus sylvestris L.) abioottisessa stressivasteessa. Polyamiinit (PAt) ovat pienimolekyylisiä typpiyhdisteitä, joiden tiedetään olevan mukana solujen perusaineenvaihdunnassa, kasvien kasvussa ja kehityksessä ja stressivasteissa. Tutkimus koostuu kolmesta kokeesta, joissa selvitettiin PA aineenvaihduntaa osana männyn taimien ja erilaistumattomien somaattisista alkioista peräisin olevien soluviljelmien stressivasteita. Kahdessa kokeellisessa tutkimuksessa männyn sirkkataimia altistettiin erilaisille kasvualustan vesipitoisuuksille ja keväthallan lämpötilaolosuhteille. Kolmannessa kokeessa PA aineenvaihdunnan reagointia osmoottiseen stressiin tutkittiin erilaistumattomissa somaattisissa epäkypsissä männyn alkiosta peräisin olevissa solukoissa kontrolloidussa nesteviljely-ympäristössä. Tutkimuksessa tarkastelin PA aineenvaihduntaa säätelevien geenien ilmenemistä ja PA pitoisuuksien muutoksia suhteessa kasvuun, kehitykseen ja stressissä indusoituvien geenien ilmenemiseen. Tulokset osoittavat, että PA aineenvaihdunta on tiukasti säädeltyä erilaisissa solukoissa. Taimien yhteyttävät solukot kerryttivät putreskiinia (Put) kuivuudessa. Ensimmäiset vasteet keväthallaolosuhteisiin havaittiin muutoksena juurten kasvun säätelyssä. Sekä kuivuus että osmoottinen stressi laskevat PA hajottavien geenien ilmenemistä, mikä on metsämännyn tapa ylläpitää tasaiset PA pitoisuudet solukoissa stressitekijöistä huolimatta. Johtopäätöksenä voidaan todeta, että PA aineenvaihdunta on tärkeä metsämännyn kasvun ja kehityksen kannalta.

Polyamines are naturally occurring cellular polycations essential for cell growth and differentiation. This investigation focused on the quantitative analysis of polyamines and metabolites in mouse erythroleukemia (MEL) cells by mass spectrometry. Hexamethylene bisacetamide (HMBA) is a synthetic polyamine derivative known to induce differentiation of a variety of transformed cells such as MEL cells. A fast and sensitive quantitative method for HMBA and metabolites NADAH, DAH and AcHA was developed using atmosphere pressure chemical ionization mass spectrometry (APCI/MS) by flow injection analysis. Selected ion monitoring (SIM) mode was employed for the mass spectrometric detection and d 4 -DAH was used as the internal standard for quantitation. The intracellular concentrations of HMBA and metabolites were obtained in MEL cells treated with 5mM HMBA in the presence or absence of 500μM APAH, a potent N 8 -acetylspermidine deacetylase inhibitor. A significant increase in intracellular NADAH and decrease in DAH levels in MEL cells were observed in HMBA treatment in the presence of APAH. This result indicates that APAH inhibits the second deacetylation step in HMBA metabolism, the conversion of NADAH to DAH, but not the first deacetylation of HMBA to NADAH. Two histone deacetylase (HDAC) inhibitors including sodium butyrate (NaB) and m -carboxycinnamic acid bis-hydroxamide (CBHA) were also used as inducing agents for MEL cell differentiation. Both agents caused accumulation of hyperacetylated histone H4 and H2B in MEL cells at concentrations optimal for inducing differentiation, while neither HMBA nor APAH had detectable effect on the acetylation level of histones. A fast and sensitive method for five important polyamines including putrescine (PU), spermidine (SPD), spermine (SPM), N 1 -acetylspermidine (N 1 -AcSPD) and N 8 -acetylspermidine (N 8 -AcSPD) was also developed using APCI/MS by flow injection analysis. Selected reaction monitoring (SRM) mode was employed for the mass spectrometric detection and 1,7-diaminoheptane was used as the internal standard for quantitation. The intracellular polyamine concentrations was obtained in MEL cells treated with 5mM HMBA, 2mM NaB, 3μM CBHA and 500μM APAH respectively. A significant increase in N 8 -acetylspermidine levels was observed during 3hr to 4 days treatment with APAH. There was no change in N 8 -acetylspermidine levels in MEL cells treated either with NaB or CBHA. The results from the present study suggest APAH has a selective inhibitory effect on N 8 -acetylspermidine but not histone deacetylation. While HDAC inhibitors inhibit histone deacetylase but have no effect on N 8 -acetylspermidine deacetylation. In conclusion, despite the known similarities they share, the enzymes involved in the deacetylation process of N 8 -acetylspermidine and histones in MEL cells are different.

Polyamines, specifically putrescine, spermidine, and spermine, are small cationic molecules found in all organisms. Cells can biosynthetically make these molecules, or alternatively, they can be transported from the extracellular environment. Malignant cells have been shown to require relatively high amounts of polyamines. There is a chemotherapeutic agent, DFMO, used to block the biosynthesis of polyamines. Many malignant cells can circumvent DFMO therapy by activating their transport system. A potential solution is to simultaneously block biosynthesis and transport of polyamines. However, little is known about the polyamine transport system in higher eukaryotes. This thesis aims to add to the basic biological understanding of the polyamine transport system, as well as contribute to our understanding of the way in which malignant cells are able to sustain rapid growth. This was done by screening six candidate genes believed to be involved in the polyamine transport system. These six genes were identified using various bioinformatics databases. They were screened using RNAi to knock down each gene of interest and by using an assay developed in our lab. One of the genes, RabX6, may play a possible role in the transport of putrescine.

La préservation des structures métalliques en milieu marin conduit au développement de revêtements organiques anticorrosion de type époxy, époxy-polyamine ou polyester, voire de nouvelles formulations respectueuses de l'environnement. Les peintures marines se trouvent exposées à la lumière solaire lors de leur utilisation en zone de marnage et elles doivent conserver leurs propriétés fonctionnelles pour assurer leur rôle de protection contre la corrosion. Afin d'évaluer le comportement à long terme de ces formulations, des vieillissements artificiels accélérés impliquant la lumière et l'eau (alternativement ou simultanément) ont été mis au point. Trois types de vieillissement ont alors été utilisés : photovieillissement à sec, cycles alternés irradiation / immersion et irradiation dans l'eau. Deux peintures marines industrielles à base de résin époxy (avec ou sans solvant) ont été étudiées ainsi que des polymères modèles : soit bidimentionnel (résine phénoxy PKHJ(R) ou tridimentionnel (DGEBA/TETA). En présence d'eau, l'étude des mécanismes de vieillissement a permis de mettre en évidence l'hydrolyse de photoproduits et leur migration dans la solution d'immersion. De plus, dans le cas de la PKHJ(R), cette hydrolyse conduit à la production de phénols qui ont un effet photoinducteur, et d'acides qui sont extraits par l'eau. Les évolutions moléculaires des matériaux ont pu être corrélées aux évolutions macroscopiques des propriétés physico-chimiques au cours du vieillissement. Les mécanismes, les cinétiques, l'influence du milieu dépendent de la nature de la structure chimique, de la structure morphologique et de l'état du plymère étudié (vieilli ou non).

O estudo do metabolismo de poliaminas (PAS), envolvendo parâmetros fisiológicos, bioquímicos e moleculares, pode gerar uma melhor compreensão do processo de maturação, e criar estratégias importantes para a otimização da embriogênese somática (ES) em cana-de-açúcar. O objetivo deste trabalho foi estudar a relação entre os conteúdos endógenos de PAs, associados à determinação da competência embriogenética e a maturação progressiva de embriões somáticos em cana-de-açúcar. Calos embriogênicos (E) e não-embriogênicos (NE) da variedade SP-803280, foram submetidos a diferentes tratamentos, utilizando agentes de maturação adicionados ao meio MS. Observou-se que as culturas NE não foram capazes de promover a diferenciação de embriões somáticos, devido principalmente, ao alto grau de oxidação das culturas. Os tratamentos suplementados com 0,75 e 1,5 g.L-1 de carvão ativado (CA), foram os que apresentaram as maiores freqüências de formação de embriões somáticos. A partir dos resultados obtidos, foi possível associar a ES em calos de cana-de-açúcar ao aumento do conteúdo endógeno de Spd (Espermidina) e Spm (Espermina), principalmente Spd, acompanhado por uma redução nos níveis de Put (Putrescina). / Somatic embryogenesis (SE) is a highly potential supplier of the material involved in the regeneration and genetic transformation of transgenic plants. The aim was to study correlations between endogenous PA contents, associated to defining embryogenetic competence and the progressive maturation of somatic embryos. Embryogenic (E) and non-embryogenic (NE) calli from the SP-803280 variety, were submitted to different maturation treatments. It was found that the NE cultures submitted to the two maturation experiments, were incapable of promoting somatic embryo differentiation, mainly due to browning. It was noted that in the first experiment, total PA endogenous content of the E callus was higher than in the NE, although, in the control, no significant differences between both callus types were encountered. We can associate the progression of sugarcane somatic embryogenesis to an increase in endogenous content of Spd e Spm, mainly Spd, accompanied for a reduction in Put levels. Thus, it was shown that somatic embryo maturation in sugarcane can be related with PA biosynthesis, thereby indicating the importance of their metabolism in the competence of sugarcane embryogenic cultures.

La sida és una malaltia causada pel VIH, que ataca a les cèl·lules del sistema immunitari. Actualment s'estima que viuen uns 33 milions de persones infectades per aquest virus. La teràpia antiretroviral actual consisteix en combinacions de dues famílies de compostos: inhibidors de la transcriptasa inversa i inhibidors de la proteasa, ambdós dirigits a enzims específics produïts pel VIH. Una de les principals dificultats en el tractament antiretroviral és la ràpida evolució del virus, que li permet fer-se resistent als fàrmacs que s'utilitzen. Per tant, hi ha una necessitat contínua d'utilitzar nous fàrmacs anti­VIH en combinació amb els actuals, i haurien d'actuar preferentment en noves dianes del cicle de replicació del VIH. El descobriment dels cofactors cel·lulars implicats en l'entrada del VIH i una millor comprensió de les proteïnes de l'envolta que controlen el procés de fusió han renovat l'interès en les primeres etapes de la replicació del VIH com a dianes per a una intervenció terapèutica. L'entrada del VIH consta de 4 etapes interrelacionades: la unió del virus a la superfície cel·lular, la unió del virus al receptor CD4, la interacció del complex CD4-glicoproteïna de l'envolta amb el coreceptors d'entrada i la fusió virus-cèl·lula. Els principals coreceptors utilitzats pel VIH per a la seva entrada a la cèl·lula són els receptors de quimiocines CCR5 i CXCR4. En aquest àmbit s'han aprovat recentment dos nous fàrmacs, Fuzeon® (Hoffman-La Roche i Trimeris), que inhibeix la fusió del virus amb la cèl·lula, i Selzentry® (Pfizer), que és un antagonista de CCR5. S'han identificat agents que bloquegen CXCR4, entre els que destaca AMD3100. Tot i això, el seu desenvolupament s'ha abandonat per una toxicitat cardíaca i falta de biodisponibilitat oral, ambdues relacionades amb la seva elevada càrrega positiva a pH fisiològic. Per tant, es necessita disposar de nous inhibidors de la unió del VIH al coreceptor CXCR4 que solucionin aquests desavantatges. L'interès d'aquesta tesi és dissenyar una quimioteca combinatòria d'anàlegs al AMD3100 amb menor basicitat, que incorporin a la seva estructura les principals característiques d'aquest compost, un linker p-fenilenbismetilènic i sistemes heterocíclics. A partir de l'estructura dels compostos de la quimioteca dissenyada es desenvolupen i validen les rutes sintètiques necessàries per a la seva obtenció. A més, s'apliquen tècniques computacionals de selecció de diversitat per a explorar l'espai químic representat pels compostos de la quimioteca. A partir de les molècules actives identificades en aquest procés i inhibidors coneguts del coreceptor CXCR4 es desenvolupen models QSAR i farmacofòrics. Aquests models s'utilitzen per a realitzar un cribatge virtual de la resta de compostos de la quimioteca dissenyada i augmentar la probabilitat de seleccionar nous compostos amb activitat antiviral. Mitjançant aquest procediment s'han identificat nous inhibidors de CXCR4, un d'ells amb elevada activitat anti-VIH. A més, aquest compost només presenta quatre àtoms de nitrogen a la seva estructura, enfront dels vuit de l'AMD3100. / El sida es una enfermedad causada por el VIH, que ataca a las células del sistema inmunitario. Actualmente se estima que viven unos 33 millones de personas infectadas por este virus. La terapia antirretroviral actual consiste en combinaciones de dos familias de compuestos: los inhibidores de la transcriptasa inversa y los inhibidores de la proteasa, ambos dirigidos a enzimas específicos producidos por el VIH. Sin embargo, una de las principales dificultades en el tratamiento antirretroviral es la rápida evolución del virus, que le permite hacerse resistente a los fármacos utilizados. Por lo tanto, hay una continua necesidad de usar nuevos fármacos anti-VIH en combinación con los actuales, que deberían actuar preferentemente en nuevas dianas del ciclo de replicación del VIH. El descubrimiento de los cofactores celulares implicados en la entrada del VIH y una mejor comprensión de las proteínas de la envoltura que controlan el proceso de fusión han renovado el interés en las primeras etapas de la replicación del VIH como dianas para una intervención terapéutica. La entrada del VIH consta de 4 etapas interrelacionadas: la unión del virus a la superficie celular, la unión del virus al receptor CD4, la interacción del complejo CD4­glicoproteína de la envoltura con los correceptores de entrada y la fusión virus-célula. Los principales correceptores utilizados por el VIH para su entrada en la célula son los receptores de quimiocinas CCR5 y CXCR4. En este ámbito se han aprobado recientemente dos nuevos fármacos, Fuzeon® (Hoffman-La Roche y Trimeris), que inhibe la fusión del virus con la célula, y Selzentry® (Pfizer), que es un antagonista de CCR5. Se han identificado agentes que bloquean CXCR4, entre los que destaca AMD3100. Sin embargo, su desarrollo se ha abandonado por una toxicidad cardiaca y falta de biodisponibilidad oral, ambas relacionadas con su elevada carga positiva a pH fisiológico. Por lo tanto se necesita disponer de nuevos inhibidores de la unión del VIH al receptor CXCR4 que solventen estas desventajas. El interés de la presente tesis es diseñar una quimioteca combinatoria de análogos al AMD3100 con menor basicidad, que incorporen en su estructura las principales características de este compuesto, un linker p­fenilenbismetilénico y sistemas heterocíclicos. A partir de la estructura de los compuestos de la quimioteca diseñada, se desarrollan y validan las rutas sintéticas necesarias para su obtención. Además, se aplican técnicas computacionales de selección de diversidad para explorar el espacio químico representado por los compuestos de la quimioteca. A partir de los compuestos activos identificados en este proceso e inhibidores conocidos del correceptor CXCR4 se desarrollan modelos QSAR y farmacofóricos. Estos modelos se utilizan para realizar un cribado virtual del resto de compuestos de la quimioteca diseñada y aumentar así la probabilidad de seleccionar nuevos compuestos con actividad antiviral. Mediante este procedimiento, se han identificado nuevos inhibidores de CXCR4, uno de ellos con elevada actividad anti-VIH. Además, este compuesto sólo presenta cuatro átomos de nitrógeno en su estructura frente a los ocho del AMD3100. / AIDS is a disease of the immune system caused by the human immunodeficiency virus (HIV). Today, about 33 million people live with this virus. Current antiretroviral therapies consist of combinations of two compound families: reverse transcriptase inhibitors and protease inhibitors, both directed to specific enzymes produced by the HIV. One of the major drawbacks in this antiretroviral treatment is drug resistance, produced by the rapid evolution of the virus. Hence there is a need to develop new anti-HIV drugs which should preferably act on new stages in HIV's life cycle. The discovery of cellular cofactors involved in the entry of HIV to host cells and a better comprehension of envelope proteins controlling the fusion process have renewed the interest in early steps of HIV replication as targets for therapeutic intervention. HIV entry to the cell can be dissected into 4 interrelated steps: virus attachment to the cell surface, virus binding to the CD4 receptor, interaction of the CD4-envelope glycoprotein complex with entry coreceptors and virus-cell fusion. The main HIV entry coreceptors are chemokine receptors CCR5 and CXCR4. In this context, two new drugs have recently been approved, Fuzeon® (Hoffman-La Roche and Trimeris), that blocks fusion of HIV with the cell, and Selzentry® (Pfizer), a CCR5 antagonist. Agents that block CXCR4 have also been identified, with AMD3100 as lead compound. However, the development of this compound has been discontinued due to cardiac toxicity and lack of oral bioavailability related to its high positive charge at physiological pH. Therefore, there is a need to develop new CXCR4 coreceptor inhibitors that will solve these limitations. The aim of this thesis is the design of a combinatorial library of AMD3100 analogues with lower basicity while preserving the main structural features of AMD3100, a p­phenylenic linker and heterocyclic systems. Synthetic routes for these compounds have been developed and validated. Moreover, computational diversity selection techniques have been applied to explore the chemical space represented by the library. Active compounds identified by this process and known CXCR4 inhibitors have been used in QSAR and pharmacophore modelling. Then, virtual screening of the rest of compounds in the library with these models has been applied to increase the probability of selecting new active compounds. This has led to the identification of new CXCR4 inhibitors, one of them showing high anti-HIV activity. Furthermore, the structure of this compound only includes four nitrogen atoms, in contrast to the eight in AMD3100.

El virus de la immunodeficiència humana de tipus 1 (VIH-1) és l'agent que provoca la síndrome de la immunodeficiència adquirida (SIDA). Una malaltia, que va ser descoberta al principi del 80, que pateixen avui en dia entre 31.1 i 35.8 milions d'individus arreu del món i que causa 2.7 milions de nous infectats cada any. Per tot això, i pels gairebé tres milions de morts que provoca cada any de mitjana aquesta malaltia, es considera important intentar bloquejar el virus (VIH-1), un paràsit que utilitza la maquinària metabòlica de la cèl·lula per replicar-se. Actualment, el tractament més comú és el TARGA (tractament antiretroviral de gran activitat), que consisteix en la combinació d'inhibidors de la transcriptasa inversa i de la proteasa, dos enzims vírics necessaris perquè es produeixi la replicació del virus. La principal problemàtica que presenta un virus d'aquestes característiques és la gran quantitat de mutacions que pot patir i, en conseqüència, la resistència que adquireix front als fàrmacs administrats. Per això és de vital importància trobar noves molècules que puguin no només suplir-ne d'altres front a aquest problema sinó que puguin interaccionar sobre noves dianes terapèutiques. No va ser fins al segle XXI que van ser aprovats fàrmacs per altres dianes terapèutiques que no fossin la transcriptasa inversa o la proteasa. Destacant-ne Fuzeon® com a inhibidor de fusió entre les membranes del VIH i la cèl·lula hoste i Selzentry® com a inhibidor de CCR5, un dels dos coreceptors d'entrada que pot emprar el virus per infectar la cèl·lula. En canvi, encara a dia d'avui no hi ha cap estructura aprovada per inhibir CXCR4, un altre coreceptor d'entrada. D'entre els compostos estudiats contra aquesta diana terapèutica en destaca l'AMD3100, un compost format per dos macrocicles (ciclam) units per un espaiador p-fenilenbismetilènic. Durant les estudis en fases clíniques, es va observar un efecte cardiotòxic i falta de biodisponibilitat oral relacionats amb l'elevada càrrega positiva a pH fisiològic. En el Laboratori de Síntesi de l'IQS, s'ha desenvolupat una família de tetraamines anàlogues del AMD3100 amb les quals, conservant l'espaiador i substituint les unitats de ciclam per diamines, s'han aconseguit bones activitats inhibidores de CXCR4. En el present treball es desenvolupen variants d'aquestes tetraamines per modificació de l'espaiador i per substitució dels ciclams per altres sistemes heterocíclics, respectivament. D'altra banda, un dels primers ITINANs (Inhibidor de la Transcriptasa Inversa No Anàleg de Nucleòsid) descoberts va ser l'1-[(2-hidroxietoxi)metil]-6-(feniltio)timina (HEPT). En el Laboratori de Disseny Molecular de l'IQS es va seleccionar una quimioteca d'anàlegs d'aquesta estructura i es va proposar una ruta sintètica. Un dels objectius d'aquest treball és doncs, a més d'obtenir anàlegs d'HEPT, confirmar sintèticament la viabilitat de l'itinerari proposat, comprovant que sigui robust, de rendiments elevats i de condicions de reacció prou suaus per poder automatitzar-lo. / El virus de la inmunodeficiencia humana de tipo 1 (VIH-1) es el agente que provoca el síndrome de la inmunodeficiencia adquirida (SIDA). Una enfermedad, que fue descubierta a principios de los 80, que sufren hoy en día entre 31.1 y 35.8 millones de individuos por todo el mundo y que causa 2.7 millones de nuevos infectados cada año. Por todo esto, y por los casi tres millones de muertos que provoca cada año de media esta enfermedad, se considera importante intentar bloquear el virus (VIH-1), un parásito que utiliza la maquinaria metabólica de la célula para replicarse. Actualmente, el tratamiento más común es el TARGA (tratamiento antirretroviral de gran actividad), que consiste en la combinación de inhibidores de la transcriptasa inversa y de la proteasa, dos enzimas víricas necesarias para que se produzca la replicación del virus. La principal problemática que presenta un virus de estas características es la gran cantidad de mutaciones que puede sufrir y, en consecuencia, la resistencia que adquiere frente a los fármacos administrados. Por eso es de vital importancia encontrar nuevas moléculas que puedan no sólo suplir a otras frente a este problema sino que puedan interaccionar sobre nuevas dianas terapéuticas. No fue hasta al siglo XXI que fueron aprobados fármacos para otras dianas terapéuticas que no fuesen la transcriptasa inversa o la proteasa. Destacan Fuzeon® como inhibidor de fusión entre membranas del VIH y la célula huésped y Selzentry® como inhibidor de CCR5, uno de los dos correceptores de entrada que puede usar el virus para infectar la célula. En cambio, aún a día de hoy no hay ninguna estructura aprobada para inhibir CXCR4, otro correceptor de entrada. De entre los compuestos estudiados contra esta diana terapéutica destaca el AMD3100, un compuesto formado por dos macrociclos (ciclam) unidos por un espaciador p-fenilenbismetilénico. Durante los estudios en fases clínicas, se observó un efecto cardiotóxico y falta de biodisponibilidad oral relacionados con la elevada carga positiva a pH fisiológico. En el Laboratorio de Síntesis del IQS, se ha desarrollado una familia de tetraaminas análogas al AMD3100 con las cuales, conservando el espaciador y sustituyendo las unidades de ciclamo por diaminas, se han conseguido buenas actividades inhibidoras de CXCR4. En el presente trabajo se desarrollan variantes de estas tetraaminas por modificación del espaciador y por sustitución de los ciclamos por otros sistemas heterocíclicos, respectivamente. Por otro lado, uno de los primeros ITINANs (Inhibidor de la Transcriptasa Inversa No Análogo de Nucleósido) descubiertos fue el 1-[(2-hidroxietoxi)metil]-6-(feniltio)timina (HEPT). En el Laboratorio de Diseño Molecular del IQS se seleccionó una quimioteca de análogos de esta estructura y se propuso una ruta sintética. Uno de los objetivos de este trabajo es pues, además de obtener análogos de HEPT, confirmar sintéticamente la viabilidad del itinerario propuesto, comprobando que sea robusto, de rendimientos elevados y de condiciones de reacción lo suficientemente suaves para poder automatizarlo. / The human immunodeficiency virus type 1 (HIV-1) is the agent which causes acquired immunodeficiency syndrome (AIDS). A disease that was discovered in the early 80s, that nowadays affects between 31.1 and 35.8 million individuals around the world and causes 2.7 million new infections each year. For all this, and for the nearly three million deaths caused each year on average by this disease, it is considered important to try to block the virus (HIV-1), a parasite which uses the cell's metabolic machinery to replicate. Currently, the most common treatment is HAART (highly active antiretroviral therapy), which is a combination of reverse transcriptase and protease inhibitors, two viral enzymes necessary for virus replication. The main issues presented by this type of virus are the large number of mutations that can occur and, therefore, the resistance it acquires to administered drugs. It is therefore of vital importance to find new molecules which can not only replace others dealing with this problem, but can interact on new therapeutic targets. It was not until the 21st century that drugs interacting with other therapeutic targets, neither reverse transcriptase nor protease, were approved. Emphasizing Fuzeon® as an inhibitor of membrane fusion between HIV and the host cell and Selzentry® as an inhibitor of CCR5, one of the two coreceptor that the virus can use to infect the cell. However, even today there is not an approved structure to inhibit CXCR4, another entry coreceptor. Among the studied compounds against this therapeutic target highlights the AMD3100, a compound formed by two macrocycles (cyclam) linked by a p-phenylenbismethylenic spacer. During clinical trials, there was a cardiotoxic effect and a lack of oral bioavailability associated with its high positive charge at physiological pH. In the Synthesis Laboratory at IQS, a family of tetraamines analogs of AMD3100 which retain the spacer and replace the cyclam units by diamines, have been developed. These compounds showed good CXCR4 inhibitory activity. In this thesis we develop variants of these tetraamines by modification of the spacer and replacement of the cyclams by other heterocyclic systems, respectively. On the other hand, one of the first NNRTIs (Non-Nucleoside Reverse Transcriptase Inhibitors) that was discovered was 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT). In the Laboratory of Molecular Design at IQS a chemical library of analogs of this structure was selected and a synthetic route was proposed. Therefore, one objective of this work is to obtain HEPT analogs, confirm the feasibility of the proposed synthetically pathway, checking the robustness of the reactions, that the yields are high and that the reaction conditions are mild enough to automate the process.