Dissertations / Theses on the topic 'Haliclona'

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico
Lectinas podem ser definidas como proteÃnas/glicoporteÃnas que reconhecem carboidratos de maneira especÃfica, mas nÃo participam do metabolismo dos mesmos e nÃo pertencem a nenhuma das principais classes de imunoglobulinas. As lectinas sÃo proteÃnas ubÃquas, estando presente em todos os organismos conhecidos. Em cÃlulas animais, lectinas tÃm sido encontradas no citoplasma, no nÃcleo e associadas a membranas das mais diversas organelas e nos mais variados tipos celulares. Tais lectinas animais podem ser classificadas em famÃlias distintas com base em suas caracterÃsticas fÃsico quÃmicas, funÃÃo e especialmente em sua identidade de estrutura primÃria e terciÃria. O objetivo deste trabalho foi purificar duas novas lectinas da esponja marinha Haliclona (Soestella) caerulea e caracterizar estruturalmente uma delas. EspÃcimes de H.caerulea foram coletados na praia do paracuru, CearÃ. Duas lectinas (H-1 e H-3) foram isoladas por tÃcnicas clÃssicas de quÃmica de proteÃnas. A estrutura primÃria de uma delas foi determinada por espectrometria de massas e RACE. A atividade tÃxica das lectinas foi avaliada frente à nÃuplios de Artemia e cepas das bactÃrias Escherichia coli e Staphylococus aureus. H-1 e H-3 apresentaram caracterÃsticas distinhas da lectina previamente isolada de H. caerulea. H-1 à uma proteÃna monomÃrica de aporoximadamente 40 kDa enquanto que H-3 à uma proteÃna trimÃrica com cadeias com massa aproximada de 9, 16 e 18 kDa. H-3 aglutina eritrÃcitos humanos do tipo A e B e foi inibida GalNAc e PSM, H-1 aglutina diversos grupos sanguineos e nÃo pÃde ser inibida por nenhum aÃÃcar testado. H-1 foi tÃxica a naÃplios de Artemia (LC50=6,4 μg.mL-1) e H-3 foi considerada nÃo tÃxica (LC50=414,2 μg.mL-1). H-3 à uma proteÃna azul, pois interage com um cromÃforo de 597 Da com absorÃÃo mÃxima a 620 nm. A estrutura primÃria de H-3 foi determinada e revelou-se Ãnica, nÃo sendo conhecida nenhuma lectina com estrutura similar. H-3 apresenta um glicano hÃbrido composto por Hex7NAcHex7DeoxiHex2. A cadeia α de H-3 sofre um processamento proteolÃtico complexo que ainda nÃo foi completamente elucidado. AlÃm disso, H-3 foi cristalizada, mas nÃo foi possÃvel a obtenÃÃo de um padraÃo de difraÃÃo que permita a resoluÃÃo da estrutura. Em suma, duas novas lectinas foram isoladas e fora observado pela primeira vez a interaÃÃo entre uma lectina e um cromÃforo natural. Pela primeira vez tambÃm, fora determinada a composiÃÃo glicÃdica de uma lectina de esponja.
Lectins are proteins/glycoproteins that recognize carbohydrate of a specific way, but not participate in the metabolism of the same and do not belong to any of major classes of immunoglobulins. Lectins are ubiquitous proteins, present in all known organisms. In animal cells, lectins have been found in the cytoplasm, in the nucleous and as membrane-associated proteins, in diverse organelles and cells. Animal lectins can be classified into distinct families based on their physicochemical characteristics, especially in their function and identity of primary and tertiary structure. The aim of this study was to purify, characterize structural and biologically new lectins from the marine sponge Haliclona (Soestella) caerulea. H. caerulea specimens were collected in Paracuru beach, CearÃ. Two lectins (H-1 and H-3) were isolated by classical techniques of protein chemistry. The primary structure of H-3 was determined by mass spectrometry and RACE. The toxic activity of lectins was evaluated against Artemia nauplii and Escherichia coli and Staphylococcus aureus strains. H-1 and H-3 showed distinct characteristics of the lectin previously isolated from H. caerulea. H-1 is a monomeric protein of 40 kDa whereas H-3 is a heterogeneus protein with chains of 9, 16 and 18 kDa. H-3 binds human erythrocytes of A and B type and was inhibited by GalNAc and PSM, H-1 binds different blood groups and could not be inhibited by any sugar tested. H-1 was toxic to Artemia nauplii (LC50 = 6.4 μg.mL-1) and H-3 was not considered toxic (LC50 = 414.2 μg.mL-1). H-3 is a blue protein that interacts with a chromophore of 597 Da of maximum absorbance at 620 nm. The primary structure of H-3 revealed a unique amino acid sequence no similar to any animal lectins known. H-3 has a hybrid glycan comprising by Hex7NAcHex7DeoxiHex2. The α-chain of H-3 undergoes complex proteolytic processing that not been fully elucidated. Moreover, H-3 was crystallized, but was not possible to obtain a diffraction pattern that permits solving the structure. In short, two new lectins were isolated and out first observed the interaction between a lectin and natural chromophore. Furthermore, for the first time given the composition glicidic out of a sponge lectin.

CARNEIRO, R. F. Lectinas da esponja marinha Haliclona (Soestella) caerulea. 2013. 127 f. Dissertação (Mestrado em Bioquímica) - Centro de Ciências, Universidade Federal do Ceará, Fortaleza, 2013.
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Lectins are proteins/glycoproteins that recognize carbohydrate of a specific way, but not participate in the metabolism of the same and do not belong to any of major classes of immunoglobulins. Lectins are ubiquitous proteins, present in all known organisms. In animal cells, lectins have been found in the cytoplasm, in the nucleous and as membrane-associated proteins, in diverse organelles and cells. Animal lectins can be classified into distinct families based on their physicochemical characteristics, especially in their function and identity of primary and tertiary structure. The aim of this study was to purify, characterize structural and biologically new lectins from the marine sponge Haliclona (Soestella) caerulea. H. caerulea specimens were collected in Paracuru beach, Ceará. Two lectins (H-1 and H-3) were isolated by classical techniques of protein chemistry. The primary structure of H-3 was determined by mass spectrometry and RACE. The toxic activity of lectins was evaluated against Artemia nauplii and Escherichia coli and Staphylococcus aureus strains. H-1 and H-3 showed distinct characteristics of the lectin previously isolated from H. caerulea. H-1 is a monomeric protein of 40 kDa whereas H-3 is a heterogeneus protein with chains of 9, 16 and 18 kDa. H-3 binds human erythrocytes of A and B type and was inhibited by GalNAc and PSM, H-1 binds different blood groups and could not be inhibited by any sugar tested. H-1 was toxic to Artemia nauplii (LC50 = 6.4 μg.mL-1) and H-3 was not considered toxic (LC50 = 414.2 μg.mL-1). H-3 is a blue protein that interacts with a chromophore of 597 Da of maximum absorbance at 620 nm. The primary structure of H-3 revealed a unique amino acid sequence no similar to any animal lectins known. H-3 has a hybrid glycan comprising by Hex7NAcHex7DeoxiHex2. The α-chain of H-3 undergoes complex proteolytic processing that not been fully elucidated. Moreover, H-3 was crystallized, but was not possible to obtain a diffraction pattern that permits solving the structure. In short, two new lectins were isolated and out first observed the interaction between a lectin and natural chromophore. Furthermore, for the first time given the composition glicidic out of a sponge lectin.
Lectinas podem ser definidas como proteínas/glicoporteínas que reconhecem carboidratos de maneira específica, mas não participam do metabolismo dos mesmos e não pertencem a nenhuma das principais classes de imunoglobulinas. As lectinas são proteínas ubíquas, estando presente em todos os organismos conhecidos. Em células animais, lectinas têm sido encontradas no citoplasma, no núcleo e associadas a membranas das mais diversas organelas e nos mais variados tipos celulares. Tais lectinas animais podem ser classificadas em famílias distintas com base em suas características físico químicas, função e especialmente em sua identidade de estrutura primária e terciária. O objetivo deste trabalho foi purificar duas novas lectinas da esponja marinha Haliclona (Soestella) caerulea e caracterizar estruturalmente uma delas. Espécimes de H.caerulea foram coletados na praia do paracuru, Ceará. Duas lectinas (H-1 e H-3) foram isoladas por técnicas clássicas de química de proteínas. A estrutura primária de uma delas foi determinada por espectrometria de massas e RACE. A atividade tóxica das lectinas foi avaliada frente à náuplios de Artemia e cepas das bactérias Escherichia coli e Staphylococus aureus. H-1 e H-3 apresentaram características distinhas da lectina previamente isolada de H. caerulea. H-1 é uma proteína monomérica de aporoximadamente 40 kDa enquanto que H-3 é uma proteína trimérica com cadeias com massa aproximada de 9, 16 e 18 kDa. H-3 aglutina eritrócitos humanos do tipo A e B e foi inibida GalNAc e PSM, H-1 aglutina diversos grupos sanguineos e não pôde ser inibida por nenhum açúcar testado. H-1 foi tóxica a naúplios de Artemia (LC50=6,4 μg.mL-1) e H-3 foi considerada não tóxica (LC50=414,2 μg.mL-1). H-3 é uma proteína azul, pois interage com um cromóforo de 597 Da com absorção máxima a 620 nm. A estrutura primária de H-3 foi determinada e revelou-se única, não sendo conhecida nenhuma lectina com estrutura similar. H-3 apresenta um glicano híbrido composto por Hex7NAcHex7DeoxiHex2. A cadeia α de H-3 sofre um processamento proteolítico complexo que ainda não foi completamente elucidado. Além disso, H-3 foi cristalizada, mas não foi possível a obtenção de um padraão de difração que permita a resolução da estrutura. Em suma, duas novas lectinas foram isoladas e fora observado pela primeira vez a interação entre uma lectina e um cromóforo natural. Pela primeira vez também, fora determinada a composição glicídica de uma lectina de esponja.

[Truncated abstract] Sponges have an invaluable ecological importance through the provision of shelter and habitat, consolidation of reefs, bio-erosion, and in benthic-pelagic coupling processes. In addition, sponges are known to be an incredible source of compounds with bio-medicinal and commercial applications. Despite their ecological and economic importance, our understanding of the processes which maintain and structure sponge populations is severely lacking compared to other sessile invertebrates (e.g. Cnidarians). This study examines the processes which help maintain and the factors which structure the populations of two sympatric Haliclona species (Demospongiae; Haplosclerida; Chalinidae) at Hamelin Bay on the south west coast of Australia. In addition, the importance of both species to the broader marine community is examined. The reproductive biology of both species was determined from histological sections taken from each species over two years at Hamelin Bay. No evidence of asexual reproduction was observed in either species. Sexual reproduction occurred from November to April in Haliclona sp. 1 (hereafter green Haliclona) and November to May in Haliclona sp. 2 (hereafter brown Haliclona). The green Haliclona is viviparous with both gonochoric and hermaphroditic individuals observed in the population. The brown Haliclona is also viviparous with separate sexes. The onset and progression of reproduction in both species corresponded to increases in water temperature and photoperiod, but only decreasing wave height showed a significant correlation to gametogenesis. ... A significant (P < 0.05) difference in concentration between seasons was also observed, suggesting environmental and physiological factors affect the production of salicylihalamide A in the green Haliclona. The importance of each species to the marine environment was assessed by investigating the endofauna inhabiting each species across their known range ( [approx. ]1000 km's). A total of 948 and 287 endofaunal individuals were found associated with the green and brown Haliclona, respectively. Twenty four endofaunal taxa were found (from mysid shrimps to teleost fish), and the endofaunal assemblages of each species were significantly different. However, only the endofaunal assemblage associated with the green Haliclona varied among locations. Overall, this study demonstrates that the populations of both species are maintained by limited sexual reproductive output and larval dispersal. Abiotic factors (e.g. water temperature, wave exposure) influence the growth and physiology of both species, which is intimately connected to their abilities to reproduce. This has important consequences for the species populations with regard to their resilience to environmental change, and potential for harvesting of biomass for supply of bioactive compounds. Additionally, both species provide important habitats for many other organisms. The findings highlight the need for a detailed understanding of the ecology of potentially exploitable sponge species, to ensure their conservation and limit the impact on the organisms which rely on the sponges.

Depuis une cinquantaine d’années, de nombreuses études ont décrit les spongiaires comme de formidables ressources de produits naturels originaux. Ces découvertes ont attisé la curiosité de nombreux scientifiques issus de domaines variés (chimie, biochimie, biologie moléculaire) qui mettent leurs connaissances en commun afin de comprendre le fonctionnement de ces animaux si particuliers. Les travaux envisagés dans ce manuscrit portent sur les éponges du genre Haliclona issu de l’ordre des Haplosclérides. En effet, ce groupe taxonomique révèle des familles de molécules très particulières, notamment les alcaloïdes 3-AP et les polyacétylènes, qui présentent un potentiel pharmacologique à exploiter. En outre la diversité chimique retrouvée au sein de ce genre remet en question la validité de la classification actuelle des Haplosclérides déjà discutée par les données de biologie moléculaire. L’intérêt particulier porté à ces composés originaux concerne également les voies métaboliques permettant d’aboutir à leur biosynthèse. Ainsi les alcaloïdes 3-AP ne trouvant aucun équivalent notamment dans le monde terrestre, la description de leur voie de biosynthèse n’a alors été envisagée que sous forme d’hypothèses. Afin d’éclaircir les différentes problématiques posées par ce groupe de spongiaires, les études menées pendant ce doctorat ont fait appel à des méthodes et techniques variées tels que les méthodes d’extraction et d’analyses de la chimie des produits naturels classique, l’outil récent qu’est la métabolomique ou encore les expériences de « feeding » et l’utilisation de composés radio-marqués
Since about fifty years, several studies described sponges as astounding resources of original natural products. These discoveries aroused scientist’s curiosity in various areas (chemistry, biochemistry, molecular biology) and to understand these animal’s functioning, they share their knowledge. The proposed work in this manuscript, focus on sponges of genus Haliclona from the order Haplosclerida. Indeed, this taxonomic group reveal peculiar chemical families, including 3-AP alkaloids and polyacetylenic compounds, displaying pharmacological potential to be exploited. Besides, chemicalDépôt de thèseDonnées complémentairesdiversity encountered in this genus challenge the actual classification of Haplosclerida group already disputed by biomolecular data. The particular interest in these original compounds also concerns metabolic pathways leading to their biosynthesis. In this way, 3-AP alkaloids, which don’t have any equivalent particularly in terrestrial area, the description of their biosynthesis pathway was only considered by hypothesis. To resolve various problematics proposed by this sponges’ group, studies conducted through this PhD appeal to several methods and technics like usual natural products chemistry extraction and analytical technics, recent tool named metabolomic or feeding experiments with radiolabelled compounds

Les travaux présentés concernent dans une première partie la synthèse biomimétique d’un alcaloïde indolomonoterpénique : la bipléiophylline. La bipléiophylline est le résultat de l’assemblage complexe de deux unités indoliques identiques ancrées sur une plateforme aromatique. Une stratégie générale de synthèse biomimétique de la bipléiophylline consistant i) à la synthèse de l’unité indolique pléiocarpamine et ii) à l’oxydation de l’acide 2,3-dihydroxybenzoïque a été envisagée. L’accès au squelette complexe de la pléiocarpamine a été étudié selon plusieurs stratégies de synthèse totale mais également par hémisynthèse. Parallèlement une étude des conditions d’oxydation notamment par électrochimie de l’acide 2,3-dihydroxybenzoïque ont permis de déterminer et caractériser son potentiel d’oxydation et de mettre au point les conditions de formation de sa forme oxydée. La seconde partie est consacrée à la synthèse biomimétique d’un modèle du cœur central de l’haliclonine A, un alcaloïde de la famille des manzamines. La synthèse de plusieurs précurseurs a été réalisée ainsi que l’étude de l’étape clé de double addition nucléophile sur un 5,6-dihydropyridinium
Our work deals in the first part with a biomimetic synthesis of bipleiophyllin, an indolomonoterpenic alkaloid. The bipleiophyllin is the result of a complex anchorage of two identical indolic subunits on an aromatic platform. A general strategy for the biomimetic synthesis of bipleiophyllin consisting of i) the synthesis of the indolic unit pleiocarpamin and ii) the oxidation of 2,3-dihydroxybenzoic acid; was considered. Access to the complex skeleton of pleiocarpamin has been studied by different total synthesis strategies but also by hemisynthesis. Meanwhile this work, a study of the oxidation conditions of 2,3-dihydroxybenzoic acid including by electrochemistry, helped identify and characterize its oxidation potential and develop the required conditions to obtain its oxidized form. The second part is devoted to the biomimetic synthesis of a model compound, mimic of the central core of haliclonin A, an alkaloid of the family of manzamins. The synthesis of several precursors and the study of the key step consisting in a double nucleophilic addition to a 5,6-dihydropyridinium were done

Dans le cadre de la recherche de nouvelles molécules antipaludiques, une espèce d'Haliclona et quatre lots de l'éponge Aplysina ianthelliformis ont été étudiées pour fournir 22 composés, dont sept sont nouveaux 141-147. Un produit connu, l'haliclonacyclamine A 129, qui a montré ici une activité in vitro sur des souches de Plasmodium falciparum FCB1 résistantes à la chloroquine avec une CI50 de 98nM et un index de sélectivité (activité Antipalsmodiale/cytotoxicité) de 57 et 67 déterminé respectivement sur cellules MVF7 et Véro. Testée in vivo sur le modèle souris, l'haliclonacyclamine A 129 a montré 48% d'inhibition de la parasitémie le 4ème jour après 3 jours de traitement à 10mg/Kg/jour administré par voie intra-péritonéale à des souris Swiss femelles infectées par P. Vinckei petteri. Les échantillons d'A. Ianthelliformis ont fourni 21 produits, tous sauf deux (aureol 137 et aplysterol 138) appartenant à la série des bromotyrosines. Ces composés 35a, 36, 130-147 montrent une faible activité in vitro sur les souches résistantes ou sensibles à la chloroquine, P. Falciparum FCB1 et 3D7 respectivement, avec des CI50 allant de 0. 9 à 44µM. Tous les composés dérivés de la bromotyrosine manquent de sélectivité, sauf l'araplysillin I 139 qui présente un faible index de sélectivité de 5,5 et 6,5 respectivement sur cellules MCF7 et Vero, et une CI50 = 4,5µM sur P. Falciparum FCB1. Tous ces produits présentent une faible activité sur la Protéinefarnésyletransférase (PFTase) ; le composé le plus actif, l'aerophobin II 135, a une CI50 = 4. 9µM. L'inactivité de la molécule 144 suggère l'importance du motif isoxazolinique dans l’activité sur PFTase
In a search for new antiplasmodial compounds, one Haliclona and four Aplysina ianthelliformis sponges were chemically investigated to furnish 21 compounds, seven of which are novel ones 141-147. The Haliclona sponge gave the known product, haliclonacylamine A 129 which exhibits potent in vitro activity against the chloroquine-resistant strain, Plasmodium falciparum FCB-1 with an IC50 of 98 nM and a strong selectivity index of 57 and 67 (determined as antiplasmodial activity/ cytotoxicity). In the in vivo mice model, haliclonacyclamine A 129 gave 45% parasitaemia inhibition on the fourth day following three days of treatment at 10 mg/Kg/day administered intra-peritoneally to Swiss female mice infected with P. Vinckei petteri. The A. Ianthelliformis sponges furnished 20 compounds, all but two (aureol 137 and aplysterol 138) of which are bromotyrosine derivatives. All bromotyrosine-derived compounds; 35a, 36, 130-147 exhibit mild in vitro activities against the chloroquine-resistant and sensitive strains, P. Falciparum FCB-1 and 3D7 respectively, with IC50s ranging between 0. 9 – 50. 5 µM. All bromotyrosine compounds lack selectivity except for araplysillin I 139 which has a weak selectivity index of 5. 5 and 6. 5 (measured for MCF-7 and Vero cells respectively) and an IC50 value of 4. 5 µM against P. Falciparum, FCB-1. All bromotyrosine derivatives, except for the novel compound 144, also exhibit weak Protein farnesyltransferase (PFTase) inhibitory activity; the most active, aerophobin II 135, has an IC50 of 8. 0 µM. The inactivity of compound 144 suggests the importance of the isoxazoline motif in conferring PFTase activity in bromotyrosine compounds

Les travaux de thèse présentés dans ce manuscrit portent sur l'étude de quatre éponges marines issues de la zone Sud-Ouest de l'Océan Indien : Plakortis kenyensis, Theonella swinhoei, Haliclona fascigera et Fascaplysinopsis reticulata. Les travaux entrepris comprenaient l'étude chimique de ces éponges incluant l'extraction, l'isolement et l'identification des métabolites secondaires par différentes techniques chromatographiques (CLMP, CLHP...) et spectroscopiques (UV-visible, HRMS, RMN 1D et 2D...). Douze métabolites secondaires ont été isolés de ces éponges dont six de structures nouvelles, à savoir : l'acide 2,5-époxydocosan-6-én-21-ynoïque (HF1), un acide gras atypique isolé de l'éponge Haliclona fascigera ; la 8-oxo-tryptamine (FR2), la 6,6’-bis-(débromo)- gelliusine F (FR3), la 6-bromo-2’-déméthyl-3’-N-méthyl-1’,8-dihydroaplysinopsine (FR6), la 5,6-dibromo-2’- déméthyl-3’-N-méthyl-1’,8-dihydroaplysinopsine (FR7) et la 5,6-dibromo-3’-déimino-2’-déméthyl-3’-oxo-1’,8- dihydroaplysinopsine (FR8), cinq alcaloïdes indoliques isolés de l'éponge Fascaplysinopsis reticulata. La valorisation des molécules isolées a ensuite été envisagée via l'évaluation de leurs activités biologiques. Parmi les douze molécules isolées, sept ont montré une activité antipaludique, trois une activité inhibitrice du quorum sensing de la bactérie bioluminescente Vibrio harveyi et cinq une activité anti-microfouling par inhibition de l'adhésion et/ou de la croissance de souches microbiennes marines
The work described in this manuscript concerns four sponges from the South-West Indian Ocean: Plakortis kenyensis, Theonella swinhoei, Haliclona fascigera and Fascaplysinopsis reticulata. The chemical study of the sponges including extraction, isolation and identification of secondary metabolites was undertaken using various chromatographic (MPLC, HPLC ...) and spectroscopic (UV-visible, HRMS, 1D and 2D NMR ...) techniques. Twelve secondary metabolites including six new molecules were isolated from these sponges. The new molecules are: 2,5-époxydocosan-6-en-21-ynoic acid (HF1) an unusual fatty acid isolated from the sponge Haliclona fascigera; 8-oxo-tryptamine (FR2), 6,6'-bis (debromo)-gelliusine F (FR3), 6-bromo-2'-demethyl-3'-N- methyl-1',8-dihydroaplysinopsine (FR6), 5,6-dibromo-2'-demethyl-3'-N-methyl-1',8-dihydroaplysinopsine (FR7) and 5,6-dibromo-3’-deimino-2’-demethyl-3’-oxo-1’,8-dihydroaplysinopsine (FR8), five indole alkaloids isolated from the sponge Fascaplysinopsis reticulata. The biological activities of the isolated molecules were then evaluated. Among the twelve isolated molecules, seven were active against the malaria parasite Plasmodium falciparum, three were identified as inhibitors of the quorum sensing-regulated bioluminescence in Vibrio harveyi and five, showing marine bacterial adhesion and/or growth inhibition, exhibited potential anti- microfouling activity

NASCIMENTO NETO, Luiz Gonzaga do. Efeito pró-cicatrizante do triterpeno 3β,6β,16β-Trihydroxylup-20(29)-ENE (CLF-1) isolado de folhas de Combretum leprosum e atividade antitumoral de uma lectina isolada da esponja marinha Haliclona caerulea. 2016. 222 f. Tese (Doutorado em biotecnologia)- Universidade Federal do Ceará, Fortaleza-CE, 2016.
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The number of cases of morbidity and mortality in patients with chronic wounds has increasing every year. In this sense, wound healing has become one the major dermatological problems. Secondary metabolites from plants have been used in several assays as promising molecules on various diseases attracted importance on treatment of various diseases. However, there are few data about it effect on wound healing. Combretum leprosum Mart. is a native species of the Caatinga and its ethanolic extract is commonly used as a healing agent, as a sedative and treatment of haemorrhages. A of the aim of the study was to evaluate the effect of triterpene 3β, 6β, 16β-trihydroxylup-20(29)-ene (CLF-1) isolated from ethanolic extract from the leaves of Combretum leprosum on skin wounds induced in vitro and in vivo lesions was avaluated. In vitro assay, the CLF-1 (2.5 μg/mL) not showed toxicity to human dermal fibroblasts (HDF). Moreover, the CLF-1 induced fibroblasts migration to the healing of the artificial injury with most effectiveness than control at 24h. In addition, the molecular mechanism of the CLF-1 treatment was studied. The results suggest that migration of fibroblasts occurs by upregulated expression of TGF-β1 and reduced levels of the inflammatory cytokine TNF-α. In the in vivo study, experimental skin lesions were created in the back of mice and treatment with the ethanolic extract from C. leprosum (EECL) and CLF-1 was evaluated for 12 days. The treatment with EECL and CLF-1 induced a faster and more effective epithelialization when compared to control. The histopathological assessment showed that EECL and CLF-1 has similar profiles during the regenerative process. This result suggest that probably the active component in the EECL may be CLF-1. Beyond of the spend about the wound healing chronic diseases as cancer are estimated as one of the main causes of dead and one of the main spending factors to the health systems. The conventional treatments, such as surgery, chemotherapy and radio are effective against primary tumors, hence not have the same effect in the later stages of the disease. In this sense, several biomolecules has attracted interest from the researchers because its antitumor potential. Marine sponjes are a biological reservoir of biomolecules, especially lectins. Lectins are proteins that bind reversibel way to carbohydrate epitops without modified them. Unlike plant lectins, there are few reports describing the mechanism of action of lectins from marine sponges to induce apoptosis in tumor cells. The effect of a lectin isolated from the marine sponge Haliclona caerulea (H3) on human breast cancer MCF-7 cell line was evaluated. H3 caused MCF7 cell reduction of viability in more than 50% (IC50=100 μg/ml) at 6h, 24h and 48h. However, on normal cells, the treatment with H3 induced a reduction in over 50% only at the highest dose tested (500 μg/ml). Furthermore, H3 provoke arrest in the G1 cell cycle phase and induce MCF7 cells apoptosis in 24h and 48h. Lysotracker Red and real-time qPCR assays suggest that effect of H3 may be related to a dynamic balance between apoptosis and autophagic cell death mediated by increase of expression of caspase-9 and LC3. In conclusion, the results suggest that CLF-1 and H3 lectin may be promising biomolecules to treatment of acute and chronic wounds and rare diseases like cancers.
O número de casos de morbimortalidade em pacientes com feridas crônicas vem aumentando a cada ano. Nesse sentido, a cicatrização de feridas tem se tornado um dos principais problemas dermatológicos. Metabólitos secundários de plantas tem sido utilizados em vários estudos como moléculas promissoras no tratamento de várias patologias. Entretanto, são poucos os dados sobre seu efeito na cicatrização de feridas. Combretum leprosum Mart. é uma planta nativa da Caatinga e seu extrato etanólico é bastante utilizado na medicina popular para cicatrização de feridas, como sedativo ou tratamento de hemorragias. Um dos objetivos do presente trabalho foi avaliar o efeito do triterpeno 3β, 6β, 16β-trihydroxylup-20 (29)-ene (CLF-1) isolado do extrato etanólico de folhas de Combretum leprosum sobre lesões induzidas in vitro e in vivo. No ensaio in vitro, o CLF-1 (2,5 μg/mL) não apresentou toxicidade a fibroblastos dermais humanos (HDF). Além disso, o CLF-1 induziu a migração de fibroblastos para o fechamento da lesão artificial de maneira mais efetiva, comparado ao controle, em 24h. Além disso, o mecanismo molecular do efeito do CLF-1 foi estudado. Os resultados sugerem que a migração de fibroblastos pode ocorrer pelo aumento da expressão de TGF-β1 e redução nos níveis da citocina inflamatória TNF-α. No estudo in vivo, lesões experimentais foram induzidas na região dorsal de camundongos e o tratamento com o extrato etanólico de C. leprosum (EECL) e CLF-1 foi avaliado por um periodo de 12 dias. Os tratamentos com EECL e CLF-1 induziram uma reepitelização mais rápida e efetiva em comparação ao controle. O estudo histopatológico mostrou que EECL e CLF-1 apresentam perfis similares durante o processo regenerativo. Esses resultados sugerem que, provavelmente o componente ativo no EECL possa ser o CLF-1. Além dos elevados custos relacionados a cicatrização de feridas, patologias crônicas como o câncer são estimados como uma das principais causas de morte e um dos principais fatores dispendiosos para os sistemas de saúde. Os tratamentos convencionais como cirurgia, quimioterapia e radioterapia são efetivos contra tumores primários, contudo, não possuem o mesmo efeito nos estágios mais avançados da doença. Nesse sentido, muitas biomoléculas tem atraído interesse da comunidade científica devido ao seu potencial antitumoral. Esponjas marinhas são consideradas reservas biológicas de várias biomoléculas, sobretudo lectinas. Lectinas são proteínas que se ligam a carboidratos de maneira reversível sem alterar sua estrutura. De modo diferente como relação a lectinas de plantas, há poucos relatos na literatura descrevendo o mecanismo de ação de lectinas de esponjas sobre a indução de apoptose de células tumorais. O efeito antitumoral de uma lectina isolada da esponja marinha Haliclona caerulea (H3) sobre células do adenocarcinoma de mama humano MCF7 foi avaliado. H3 induziu a redução da viabilidade celular de MCF7 em mais de 50% (IC50=100 μg/mL) em 6h, 24h e 48h. Contudo, sobre células normais, o tratamento com H3 induziu a redução em mais de 50%, apenas na maior dose testada (500 μg/mL). Além disso, H3 provoca arraste no ciclo celular na fase G1 e induz apoptose das células MCF7 em 24 e 48h. Ensaios utilizando Lysotracker Red e PCR em tempo-real sugerem que o efeito de H3 pode estar relacionado a um balanço dinâmico entre apoptose e autofagia, mediados pelo aumento da expressão de caspase-9 e LC3. Em conclusão, os resultados sugerem que o CLF-1 e H3 podem ser biomoléculas promissoras para o tratamento de feridas agudas e crônicas e enfermidades como câncer.

CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior
The number of cases of morbidity and mortality in patients with chronic wounds has increasing every year. In this sense, wound healing has become one the major dermatological problems. Secondary metabolites from plants have been used in several assays as promising molecules on various diseases attracted importance on treatment of various diseases. However, there are few data about it effect on wound healing. Combretum leprosum Mart. is a native species of the Caatinga and its ethanolic extract is commonly used as a healing agent, as a sedative and treatment of haemorrhages. A of the aim of the study was to evaluate the effect of triterpene 3β, 6β, 16β-trihydroxylup-20(29)-ene (CLF-1) isolated from ethanolic extract from the leaves of Combretum leprosum on skin wounds induced in vitro and in vivo lesions was avaluated. In vitro assay, the CLF-1 (2.5 μg/mL) not showed toxicity to human dermal fibroblasts (HDF). Moreover, the CLF-1 induced fibroblasts migration to the healing of the artificial injury with most effectiveness than control at 24h. In addition, the molecular mechanism of the CLF-1 treatment was studied. The results suggest that migration of fibroblasts occurs by upregulated expression of TGF-β1 and reduced levels of the inflammatory cytokine TNF-α. In the in vivo study, experimental skin lesions were created in the back of mice and treatment with the ethanolic extract from C. leprosum (EECL) and CLF-1 was evaluated for 12 days. The treatment with EECL and CLF-1 induced a faster and more effective epithelialization when compared to control. The histopathological assessment showed that EECL and CLF-1 has similar profiles during the regenerative process. This result suggest that probably the active component in the EECL may be CLF-1. Beyond of the spend about the wound healing chronic diseases as cancer are estimated as one of the main causes of dead and one of the main spending factors to the health systems. The conventional treatments, such as surgery, chemotherapy and radio are effective against primary tumors, hence not have the same effect in the later stages of the disease. In this sense, several biomolecules has attracted interest from the researchers because its antitumor potential. Marine sponjes are a biological reservoir of biomolecules, especially lectins. Lectins are proteins that bind reversibel way to carbohydrate epitops without modified them. Unlike plant lectins, there are few reports describing the mechanism of action of lectins from marine sponges to induce apoptosis in tumor cells. The effect of a lectin isolated from the marine sponge Haliclona caerulea (H3) on human breast cancer MCF-7 cell line was evaluated. H3 caused MCF7 cell reduction of viability in more than 50% (IC50=100 μg/ml) at 6h, 24h and 48h. However, on normal cells, the treatment with H3 induced a reduction in over 50% only at the highest dose tested (500 μg/ml). Furthermore, H3 provoke arrest in the G1 cell cycle phase and induce MCF7 cells apoptosis in 24h and 48h. Lysotracker Red and real-time qPCR assays suggest that effect of H3 may be related to a dynamic balance between apoptosis and autophagic cell death mediated by increase of expression of caspase-9 and LC3. In conclusion, the results suggest that CLF-1 and H3 lectin may be promising biomolecules to treatment of acute and chronic wounds and rare diseases like cancers.
O nÃmero de casos de morbimortalidade em pacientes com feridas crÃnicas vem aumentando a cada ano. Nesse sentido, a cicatrizaÃÃo de feridas tem se tornado um dos principais problemas dermatolÃgicos. MetabÃlitos secundÃrios de plantas tem sido utilizados em vÃrios estudos como molÃculas promissoras no tratamento de vÃrias patologias. Entretanto, sÃo poucos os dados sobre seu efeito na cicatrizaÃÃo de feridas. Combretum leprosum Mart. à uma planta nativa da Caatinga e seu extrato etanÃlico à bastante utilizado na medicina popular para cicatrizaÃÃo de feridas, como sedativo ou tratamento de hemorragias. Um dos objetivos do presente trabalho foi avaliar o efeito do triterpeno 3β, 6β, 16β-trihydroxylup-20 (29)-ene (CLF-1) isolado do extrato etanÃlico de folhas de Combretum leprosum sobre lesÃes induzidas in vitro e in vivo. No ensaio in vitro, o CLF-1 (2,5 μg/mL) nÃo apresentou toxicidade a fibroblastos dermais humanos (HDF). AlÃm disso, o CLF-1 induziu a migraÃÃo de fibroblastos para o fechamento da lesÃo artificial de maneira mais efetiva, comparado ao controle, em 24h. AlÃm disso, o mecanismo molecular do efeito do CLF-1 foi estudado. Os resultados sugerem que a migraÃÃo de fibroblastos pode ocorrer pelo aumento da expressÃo de TGF-β1 e reduÃÃo nos nÃveis da citocina inflamatÃria TNF-α. No estudo in vivo, lesÃes experimentais foram induzidas na regiÃo dorsal de camundongos e o tratamento com o extrato etanÃlico de C. leprosum (EECL) e CLF-1 foi avaliado por um periodo de 12 dias. Os tratamentos com EECL e CLF-1 induziram uma reepitelizaÃÃo mais rÃpida e efetiva em comparaÃÃo ao controle. O estudo histopatolÃgico mostrou que EECL e CLF-1 apresentam perfis similares durante o processo regenerativo. Esses resultados sugerem que, provavelmente o componente ativo no EECL possa ser o CLF-1. AlÃm dos elevados custos relacionados a cicatrizaÃÃo de feridas, patologias crÃnicas como o cÃncer sÃo estimados como uma das principais causas de morte e um dos principais fatores dispendiosos para os sistemas de saÃde. Os tratamentos convencionais como cirurgia, quimioterapia e radioterapia sÃo efetivos contra tumores primÃrios, contudo, nÃo possuem o mesmo efeito nos estÃgios mais avanÃados da doenÃa. Nesse sentido, muitas biomolÃculas tem atraÃdo interesse da comunidade cientÃfica devido ao seu potencial antitumoral. Esponjas marinhas sÃo consideradas reservas biolÃgicas de vÃrias biomolÃculas, sobretudo lectinas. Lectinas sÃo proteÃnas que se ligam a carboidratos de maneira reversÃvel sem alterar sua estrutura. De modo diferente como relaÃÃo a lectinas de plantas, hà poucos relatos na literatura descrevendo o mecanismo de aÃÃo de lectinas de esponjas sobre a induÃÃo de apoptose de cÃlulas tumorais. O efeito antitumoral de uma lectina isolada da esponja marinha Haliclona caerulea (H3) sobre cÃlulas do adenocarcinoma de mama humano MCF7 foi avaliado. H3 induziu a reduÃÃo da viabilidade celular de MCF7 em mais de 50% (IC50=100 μg/mL) em 6h, 24h e 48h. Contudo, sobre cÃlulas normais, o tratamento com H3 induziu a reduÃÃo em mais de 50%, apenas na maior dose testada (500 μg/mL). AlÃm disso, H3 provoca arraste no ciclo celular na fase G1 e induz apoptose das cÃlulas MCF7 em 24 e 48h. Ensaios utilizando Lysotracker Red e PCR em tempo-real sugerem que o efeito de H3 pode estar relacionado a um balanÃo dinÃmico entre apoptose e autofagia, mediados pelo aumento da expressÃo de caspase-9 e LC3. Em conclusÃo, os resultados sugerem que o CLF-1 e H3 podem ser biomolÃculas promissoras para o tratamento de feridas agudas e crÃnicas e enfermidades como cÃncer.

A descoberta de fármacos a partir de produtos isolados de organismos marinhos tem apresentado um grande crescimento nos últimos anos, principalmente devido aos avanços tecnológicos analíticos, síntese química e biotecnologia. Dentre estes, as esponjas representam uma das principais fontes de metabólitos protótipos para diversas atividades, destacando-se os efeitos antitumorais. Neste contexto, este trabalho teve como objetivo a investigação química e biológica de três esponjas coletadas na costa sul-brasileira: Haliclona tubifera, Polymastia janeirensis e Scopalina ruetzleri. Considerando a correlação entre câncer, distúrbios da coagulação e desbalanço de espécies reativas de oxigênio (EROs), foram realizados ensaios visando a aquisição destas atividades e a identificação de substâncias bioativas. Para a esponja H. tubifera foram observados interessantes efeitos antitumorais em células de glioma e neuroblastoma humano (IC50 < 20 μg/ml), além das atividades antioxidante e anticoagulante para a fração acetato de etila. O composto majoritário desta fração foi isolado como um derivado N-Boc e sua configuração foi estabelecida utilizando um novo protocolo de dicroísmo circular e semissíntese de derivados. Assim, este esfingosídeo de cadeia longa isolado (2R,3R,6R,7Z)-2-amino-7-octadecene-1,3,6-triol, foi denominado halisphingosine A. Um novo composto minoritário, halisphingosine B foi obtido usando técnicas de isolamento em escala nanomolar. Sua configuração absoluta foi estabelecida por comparação com o composto A. Da mesma forma, para a esponja S. ruetzleri, a fração acetato de etila demonstrou os resultados mais promissores. Um potencial efeito anticâncer e de inibição dos radicais peroxila foi observado. Além disso, um efeito modulador da peroxidação lipídica foi evidenciado em ensaio ex vivo de dienos conjugados. Através da análise por RMN de 1H, verificou-se que a fração era majoritariamente constituída por ácidos graxos, os quais foram derivatizados para caracterização por Cromatografia Gasosa (GC/FID). Foram identificados 32 ácidos graxos principalmente poli-insaturados (53%). Ácidos graxos minoritários não usuais para o ambiente marinho também foram caracterizados. A esponja P. janeirensis apresentou os efeitos citotóxicos mais promissores em células de glioma e neuroblastoma humano, com um IC50 < 1,0 μg/ml para o extrato aquoso (pH 6,8), sendo este efeito pH-dependente, uma vez que o extrato (pH 5,8) não alterou a viabilidade celular. Para P. janeirensis, foi também investigado seu potencial de defesa química em modelo de Zebrafish. Foi observado que o extrato aquoso desencadeia um efeito de fuga, alterando significativamente o comportamento espaço-temporal de peixes Danio rerio. Analisando em conjunto, os dados do presente trabalho representam uma nova contribuição para o estudo químico e biológico de espécies de esponjas marinhas da costa sul-brasileira e apontam as potencialidades destas esponjas na busca de moléculas protótipos para fármacos, especialmente relacionados à terapia do câncer.
Drug discovery from marine natural products has increased in the past few years, mainly due to technological advances in spectroscopy, chemical synthesis and biotechnology. Among all marine animals, sponges represent one of the major sources of prototype metabolites for several biological activities, highlighting the antitumor effects. In this context, this study carried out chemical and biological investigation of three sponges collected on the South Brazilian coastline: Haliclona tubifera, Polymastia janeirensis and Scopalina ruetzleri. Considering the correlation between cancer, clotting disorders and imbalance of reactive oxygen species (ROS), experiments were conducted for acquisition of these activities and identification of bioactive compounds. H. tubifera showed an interesting cytotoxic effect in human neuroblastoma and glioma cell lines (IC50 <20 μg/ml), antioxidant and anticoagulant effect for ethyl acetate (EtOAc) fraction. The major compound of EtOAc fraction was isolated as an N-Boc derivative and its configuration was established using a new circular dichroism protocol with the production of semi-synthetic derivatives. This long chain sphingoid base (2R,3R,6R,Z)-2-aminooctadec-7-ene-1,3,6-triol was named as halisphingosine A. A new minor compound, halisphingosine B was obtained using nanomol scale techniques and their absolute configuration was established by comparison with compound A. Likewise, for the sponge S. ruetzleri, the EtOAc fraction showed the most promising results. A potential anticancer effect, inhibition of peroxyl radicals and modulation effect of lipid peroxidation was observed. Fingerprint 1H NMR analysis showed that this fraction is mainly constituted of fatty acids. Through Fatty Acid Methyl Ester (FAMEs) analysis by GC/FID, it was possible to identify 32 fatty acids, of which around 50% were Polyunsaturated Fatty Acids (PUFAs). In addition, some minor unusual fatty acids for the marine biosphere were identified. It was observed for P. janeirensis the most promising cytotoxic effects on human glioma and neuroblastoma cells, with an IC50 <1.0 μg/ml to aqueous extract (pH 6.8), being this effect pH-dependent, since the extract (pH 5.8) did not affect the cell viability. Moreover, P. janeirensis was investigated along their potential chemical defense in Zebrafish model. Aqueous extract trigged an escape effect, significantly altering the spatio-temporal swimming activity of animals. Taken together, the data presented from this study represent a new contribution to chemical and biological research of marine sponge species from South Brazilian coastline, and point the potentialities of sponges to search chemical prototypes for drugs, especially related to cancer therapy.

Orientador: Ronaldo Aloise Pilli
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Quimica
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Resumo: Os alcalóides marinhos haliclorina (1), ácido pináico (2) e ácido tauropináico (3), isolados, por D. Uemura e colaboradores em 1996, apresentam em comum um sistema 6-aza-[4.5.0]-espirobiciclodecano. A atividade biológica da haliclorina (1) está relacionada com a inibição de moléculas associadas à adesão de células vasculares (VCAM-1) com IC50 de 7mg/mL. O ácido pináico (2) e ácido tauropináico (3) são inibidores da fosfolipase A2 (FLA2). Devido à similaridade estrutural existente entre haliclorina (1), ácido pináico (2) e ácido tauropináico (3), a proposta sintética para estes produtos naturais apresenta um intermediário chave em comum, o núcleo 6-aza-[4.5.0]- espirobiciclodecano. A estratégia sintética foi baseada em uma reação de Michael estereosseletiva entre enolato de lítio da N-propionilpirolidina e 1-ciclopenten-1-carboxilato de metila, seguida da alquilação in situ com 4-iodo-butirato de etila formando 4 em 68% rendimento. A próxima etapa consistiu na condensação de Dieckmann seguida de hidrólise/descarboxilação conduzindo a cetona 5 (61% rendimento) que sofreu redução com LiEt3BH, seguida de lactonização espontânea para gerar 6 (67% rendimento). Após algumas manipulações de grupo funcionais foi obtida a oxima 7 (76% rendimento de 6) precursora do rearranjo de Beckmann que forneceu a lactama espirobicíclica 8 em 60% rendimento
Abstract: In 1996, D. Uemura and co-workers isolated the marine alkaloids halichlorine (1), pinnaic acid (2) and tauropinaic acid (3). They are structurally co-related by a 6- azaspiro[4.5.0]decane core. The biological activity of the haliclorina (1) is related to the inhibition of molecules associated to the adhesion of vascular cells (VCAM-1) with IC50 7mg/mL. The pinnaic acid (2) and tauropinnaic acid (3) are inhibitors of the fosfolipase A2 (FLA2). Due to the structural similarity among halichlorine (1), acid pinnaic (2) and acid tauropinnaic (3), this work presents a new synthetic approach to a common key intermediate, the 6-azaspiro[4.5.0]decane nucleus. Our approach was based on the tandem Michael addition/alkylation of the lithium enolate of N-propionyl pyrrolidine to 1-carbomethoxy cyclopentene, followed by in situ alkylation with ethyl 4-iodobutanoate to provide 4 in 68% yield. Dieckmann cyclization, followed by decarboxylation, afforded spirobicyclic ketone 5 (61% yield) which underwent reduction with LiEt3BH reduction, followed by spontaneous lactonization to give 6 (67% yield). Straightforward functional group manipulations provided oxime 7 (76% yield from 6) which underwent Beckmann rearrangement to afford the spirobicyclic lactam 8 in 60% yield, a potential intermediate to the synthesis of those alkaloids
Doutorado
Quimica Organica
Doutor em Ciências

Thesis (M.S.)--Florida State University, 2003.
Advisor: Dr. Don Levitan, Florida State University, College of Arts and Sciences, Dept. of Biological Science. Title and description from dissertation home page (viewed Apr. 7, 2004). Includes bibliographical references.