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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
691

Contribution à l'étude phytochimique de Solidago virgaurea : application dans le domaine bucco-dentaire et étude de la variabilité phytochimique pour la création d'une filière / Contribution to the phytochemical study of Solidago virgaurea

Laurençon, Lise 12 April 2013 (has links)
Dans le but de valoriser la biodiversité végétale des Alpes-Maritimes, une plante commune dans cette région, Solidago virgaurea, a été sélectionnée pour son potentiel inhibiteur de la conversion levure-hyphe de Candida albicans, micro-organisme responsable d’infections bucco-dentaires de type candidose. Le fractionnement bioguidé de l’extrait aqueux a conduit à l’identification d’une famille de saponines particulièrement active. Parmi les onze saponines majoritaires caractérisées par RMN et HRMS, cinq se sont révélées être de nouvelles structures. Les tests biologiques ont néanmoins montré qu’elles n’étaient pas toutes actives contre la forme filamenteuse de C. albicans. Ces résultats ont conduit à une étude de la variabilité de la composition en saponines de plusieurs populations alpines de S. virgaurea. Trois méthodes de dosage des saponines ont été développées par HPLC et HPTLC. Les résultats ont démontré l'influence de différents facteurs sur la composition en saponines. Enfin, la composition globale de différents extraits de S. virgaurea a été étudiée dans le but d'identifier des activités biologiques complémentaires. Parmi les composés identifiés, trois nouveaux acides octulosoniques ont été caractérisés, aux côtés de trois composés phénoliques identifiés pour la première fois chez S. virgaurea. Les tests biologiques sur les extraits et fractions ont par ailleurs mis en évidence des activités antioxydante, anti-tyrosinase et inhibitrice de cellules cancéreuses in vitro. Ces tests devront être approfondis ultérieurement. / Toward the promotion of plant diversity of Maritime Alps, a common plant of the alpine area, Solidago virgaurea, was chosen to its inhibiting activity of Candida albicans yeast-hyphal conversion, a causal agent of opportunistic oral infections named candidiasis. In a first step, an aqueous extract of S. virgaurea was submitted to bioassay guided fractionation. This led to an active saponin-containing fraction from which eleven saponins were characterized by carrying out NMR experiments along with HRMS analyses. Five out of these were identified for the first time and bioassays showed that saponins activity varied according to the molecular structure of the compound. In a second step, the saponins composition of various S. virgaurea populations was studied qualitatively and quantitatively, using HPLC and HPTLC. Results demonstrated that saponins composition depends on various factors. Finally, the overall chemical composition of different S. virgaurea extracts was investigated searching for additional bioactivities. Among all the identified compounds, three new octulosonic acids were characterized and three phenolic compounds were found for the first time in S. virgaurea. Moreover, bioassays on extracts and fractions showed antioxidant, anti-tyrosinase activity and inhibition of cancer cell lines in vitro. Further bioassays have now to be completed. As a conclusion, this work was the starting point of an oral care product development and the setting-up of an innovative sector.
692

MECANISMOS DE TOXICIDADE DE Duguetia furfuraceae A. St.-Hill NO MODELO DE Drosophila melanogaster E AVALIAÇÃO DO POTENCIAL ANTIFÚNGICO. / MECHANISMS OF TOXICITY OF Duguetia furfuracea A. St.-Hil. IN Drosophila melanogaster AND EVALUATION OF ITS ANTIFUNGAL POTENTIAL

Pinho, Francisca Valéria Soares de Araújo 04 December 2015 (has links)
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Duguetia furfuracea is a common shrub from Brazilian cerrado areas, known as "ata brava", often used as a medicinal plant especially in treatment of renal colic and rheumatism. However, pharmacological studies with extracts obtained from different parts of this plant have shown cytotoxic activity, bactericidal and anti-tumor. Thus, this study aimed to identify and quantify the phenolic compounds of the hydroalcoholic extract of leaves D. furfuracea (HEDP), methanol (Mt-OH) and ethyl acetate (Ac-OEt) fractions by HPLC, and carry out phytochemical screening for different classes of compounds. In addition the antioxidant activity by the DPPH and FRAP assays, toxicity of the crude extract in the Drosophila melanogaster model, and the antifungal/modulatory activity were evaluated. The phytochemical screening revealed the presence of alkaloids, tannins, xanthones, chalcones, flavonoids, aurones and phenolic acids. HPLC analysis revealed that major components of HEDF were caffeic acid (33.17 ± 0.03 mg/g) and rutin (20.56 ± 0.01 mg/g), for the Mt-OH fraction were, caffeic acid (32.47 ± 0.03 mg/g) and quercitrin (31.96 ± 0.03 mg/g), and for Ac-OEt fraction were, quercitrin (32.97 ± 0.03 mg/g) and isoquercitrin (31.56 ± 0.01 mg/g). Although highest levels of phenols and total flavonoids were found in Ac-OEt fraction, the crude extract showed the highest antioxidant (in vitro) potential. The toxicity of the extract was confirmed (in vivo) associating the extract with standard diet of D. melanogaster at different concentrations. The extract caused a significant increase in mortality on the third day of exposure of flies at higher concentrations (100 and 200 mg/ml). The 50mg/ml concentration promoted decrease in motility of flies. Interestingly, the activity of acetylcholinesterase (AchE) was increased in flies exposed to concentrations of 1 and 10mg/ml and inhibited by the concentration of 50 mg/ml of HEDF. The cell viability was significantly compromised flies at all HEDF concentrations (1, 10 and 50 mg/ml) although it has been observed a significant increase in production of reactive oxygen species flies exposed only in the concentration of 50mg/ml. In this context it was evaluated the influence of extract (ex vivo) in the activity of antioxidant defenses of D. melanogaster exposed to concentrations of 1 and 10 mg/ml of HEDF resulted in a significant increase in the activity of enzymes Glutathione s-transferase (GST), Superoxide dismutase (SOD) and catalase (CAT), however, for the concentration of 50 mg/ml of extract, there was a dramatic decrease in the activity of GST with no change in SOD activity and CAT. The cleavage of PARP investigated as a general index of cell death by apoptosis was confirmed in flies exposed to all HEDF concentrations. Yet exposure of the flies to HEDF 10 mg/mL significantly increased the phosphorylation of kinase ERK. The extract and fractions tested against Candida albicans, Candida tropicalis and Candida krusei showed no fungicidal activity since the minimum inhibitory concentration (MIC) was ≥ 1.024 μg/ml for all fungal strains tested. However, the HEDF and the fractions Ac-OEt and Mt-OH had a synergistic effect when combined with fluconazole, indicating modulatory action against fungi when associated with clinically relevant medicine. The HEDF and the fraction Mt-OH potentiated the effect of fluconazole when tested against C. kruzei, and the fraction Mt-OH also showed synergy with fluconazol against C. tropicalis. The fraction Ac-OEt potentiated the effect of fluconazol against C. albicans. The set of results suggests that oxidative stress may be an important mechanism underlying the toxicity induced by extract of D. furfuracea in Drosophila melanogaster. Additionally modulatory activity of the extract and fractions towards fluconazol improve the biomedical applications D. furfuracea. / Duguetia furfuracea é um arbusto comum em áreas do cerrado brasileiro, conhecida como ata brava , costuma ser usada como planta medicinal especialmente no combate a cólica renal e reumatismo. Entretanto, estudos farmacológicos com extratos obtidos de diferentes partes dessa planta têm evidenciado atividades citotóxica, bactericida e antitumoral. Assim, esse trabalho teve como objetivos identificar e quantificar os compostos fenólicos do extrato hidroalcoólico das folhas de Duguetia furfuracea (EHDF) e frações metanólica (Mt-OH) e acetato de etila (Ac-OEt) por meio de HPLC, e realizar triagem fitoquímica para diferentes classes de compostos. Ainda avaliar a atividade antioxidante por meio dos ensaios DPPH e FRAP, a toxicidade do extrato bruto no modelo de Drosophila melanogaster, a atividade antifúngica e modulatória do extrato e frações. A triagem fitoquímica revelou presença de alcalóides, taninos, xantonas, chalconas, flavonoides, auronas e ácidos fenólicos. A análise por HPLC no EHDF revelou como principais componentes ácido caféico (33,17 ± 0,03 mg/g) e rutina (20,56 ± 0,01 mg/g), para a fração Mt-OH, o ácido caféico (32,47 ± 0,03 mg/g) e quercitrina (31,96 ± 0,03 mg/g), e para Ac-OEt, quercitrina (32,97 ± 0,03 mg/g) e isoquercitrina (31,56 ± 0,01 mg/g). Os mais altos níveis de fenois e flavonoides totais foram encontrados na fração Ac-OEt, entretanto, o extrato bruto apresentou maior poder antioxidante (in vitro) do que as frações. A toxicidade do extrato foi confirmada no modelo in vivo Drosophila melanogaster. O extrato promoveu aumento significativo da mortalidade no terceiro dia de exposição das moscas nas concentrações mais altas (100 e 200mg/ml). A concentração de 50mg/ml promoveu redução na motilidade das moscas. A atividade da acetilcolinesterase (AchE) foi aumentada nas moscas expostas as concentrações de 1 e 10 mg/ml e inibida na concentração de 50 mg/ml do EHDF. A viabilidade celular das moscas foi significativamente comprometida em todas as concentrações do EHDF (1; 10 e 50mg/ml) embora tenha sido observado um aumento significativo na produção de espécies reativas de oxigênio apenas nas moscas expostas a concentração de 50mg/ml. Nesse contexto foram avaliadas as defesas antioxidantes de D. melanogaster A exposição das moscas as concentrações de 1 e 10 mg/ml do HEDF resultou em aumento significativo na atividade das enzimas Glutationa s-transferase (GST), Superóxido dismutase (SOD) e Catalase (CAT), entretanto, na concentração de 50 mg/ml do extrato, houve diminuição na atividade da GST sem alterações na atividade da SOD e CAT. A clivagem da PARP, investigada como índice geral de morte celular por apoptose, foi confirmada nas moscas expostas a todas as concentrações do EHDF. Ainda a exposição das moscas a 10 mg/mL do EHDF aumentou significativamente a fosforilação de ERK. O extrato e frações testados contra Candida albicans, Candida tropicalis e Candida krusei não apresentaram atividade fungicida considerando que a concentração inibitória mínima (CIM) foi ≥ 1,024 μg/ml para todas as estirpes de fungos testadas. Entretanto, o extrato e as frações Ac-OEt e Mt-OH apresentaram efeito sinérgico quando associadas ao fluconazol, indicando ação moduladora contra fungos mediante associação a medicamento clinicamente relevantes. O EHDF e a fração Mt-OH potencializaram o efeito do fluconazol quando testados contra a C. kruzei, e a fração Mt-OH também apresentou sinergismo com fluconazol contra C. tropicalis. A fração Ac-OEt potencializou o efeito do fluconazol contra C. albicans. O conjunto dos resultados sugere que o estresse oxidativo pode ser um mecanismo importante subjacente a toxicidade induzida por Duguetia furfuracea em Drosophila melanogaster, e atividade modulatória do extrato e frações junto ao fluconazole amplia as aplicações biomédicas de Duguetia furfuracea.
693

Effect of fatty acids on hyphal growth in the pathogenic yeast Candida albicans

Shareck, Julie 09 1900 (has links)
Candida albicans est une levure pathogène qui, à l’état commensal, colonise les muqueuses de la cavité orale et du tractus gastro-intestinal. De nature opportuniste, C. albicans cause de nombreuses infections, allant des candidoses superficielles (muguet buccal, vulvo-vaginite) aux candidoses systémiques sévères. C. albicans a la capacité de se développer sous diverses morphologies, telles que les formes levures, pseudohyphes et hyphes. Des stimuli environnementaux mimant les conditions retrouvées chez l’hôte (température de 37°C, pH neutre, présence de sérum) induisent la transition levure-à-hyphe (i.e. morphogenèse ou filamentation). Cette transition morphologique contribue à la pathogénicité de C. albicans, du fait que des souches présentant un défaut de filamentation sont avirulentes. Non seulement la morphogenèse est un facteur de virulence, mais elle constituerait aussi une cible pour le développement d’antifongiques. En effet, il a déjà été démontré que l’inhibition de la transition levure-à-hyphe atténuait la virulence de C. albicans lors d’infections systémiques. Par ailleurs, des études ont démontré que de nombreuses molécules pouvaient moduler la morphogenèse. Parmi ces molécules, certains acides gras, dont l’acide linoléique conjugué (CLA), inhibent la formation d’hyphes. Ainsi, le CLA posséderait des propriétés thérapeutiques, du fait qu’il interfère avec un déterminant de pathogénicité de C. albicans. Par contre, avant d’évaluer son potentiel thérapeutique dans un contexte clinique, il est essentiel d’étudier son mode d’action. Ce projet vise à caractériser l’activité anti-filamentation des acides gras et du CLA et à déterminer le mécanisme par lequel ces molécules inhibent la morphogenèse chez C. albicans. Des analyses transcriptomiques globales ont été effectuées afin d’obtenir le profil transcriptionnel de la réponse de C. albicans au CLA. L’acide gras a entraîné une baisse des niveaux d’expression de gènes encodant des protéines hyphes-spécifiques et des régulateurs de morphogenèse, dont RAS1. Ce gène code pour la GTPase Ras1p, une protéine membranaire de signalisation qui joue un rôle important dans la transition levure-à-hyphe. Des analyses de PCR quantitatif ont confirmé que le CLA inhibait l’induction de RAS1. De plus, le CLA a non seulement causé une baisse des niveaux cellulaires de Ras1p, mais a aussi entraîné sa délocalisation de la membrane plasmique. En affectant les niveaux et la localisation cellulaire de Ras1p, le CLA nuit à l’activation de la voie de signalisation Ras1p-dépendante, inhibant ainsi la morphogenèse. Il est possible que le CLA altère la structure de la membrane plasmique et affecte indirectement la localisation membranaire de Ras1p. Ces travaux ont permis de mettre en évidence le mode d’action du CLA. Le potentiel thérapeutique du CLA pourrait maintenant être évalué dans un contexte d’infection, permettant ainsi de vérifier qu’une telle approche constitue véritablement une stratégie pour le traitement des candidoses. / The yeast Candida albicans is an inhabitant of the oral cavity, the gastrointestinal and genitourinary tracts of humans. Generally encountered as a commensal, it is also an opportunistic pathogen that causes a spectrum of infections, ranging from superficial mycoses (thrush, vulvovaginitis) to severe and life-threatening systemic infections. A striking feature of C. albicans is its ability to grow in different morphological forms, including budding yeasts, pseudohyphae, and hyphae. Environmental cues that mimic host conditions (elevated temperature, neutral or alkaline pH, and serum) induce the yeast-to-hypha transition. Morphogenesis is considered to be an attribute of pathogenesis, as mutants locked as yeasts or filamentous forms are avirulent. Given that the yeast-to-hypha transition is a virulence factor, it may also constitute a target for the development of antifungal drugs. Indeed, evidence has shown that impairing morphogenesis is a means to treat systemic candidiasis. Concurrently, a number of molecules have been reported to modulate morphogenesis in C. albicans. For instance, several fatty acids, including conjugated linoleic acid (CLA), inhibited the yeast-to-hypha transition. By interfering with an important attribute of C. albicans pathogenesis, CLA may harbor antifungal properties. However, before assessing its therapeutic potential in a clinical context, it is mandatory to address CLA’s mode of action. The present study aims to further characterize the hypha-inhibiting properties of fatty acids and CLA and to elucidate the mechanism by which these molecules inhibit the yeast-to-hypha transition in C. albicans. Gene expression analyses were performed to gain insight into the transcriptional response of cells to CLA on a genome-wide scale and to probe the fatty acid’s mode of action. CLA downregulated the expression of hypha-specific genes and blocked the induction of genes encoding regulators of hyphal growth, including that of RAS1, which encodes the small GTPase Ras1p. A membrane-associated signaling protein, Ras1p plays a major role in morphogenesis. Quantitative PCR analyses showed that CLA prevented the increase in RAS1 mRNA levels which occurred at the onset of the yeast-to-hypha transition. Unexpectedly, CLA reduced the steady-state levels of Ras1p. Additionally, CLA caused the delocalization of GFP-Ras1p from the plasma membrane. These findings indicate that CLA treatment results in suboptimal Ras1p cellular concentrations and localization, which impedes Ras1p signaling and inhibits the yeast-to-hypha transition. CLA may indirectly affect Ras1p localization by altering the structure of the plasma membrane. These studies have provided the mechanism underlying CLA’s hypha-inhibiting properties and may serve as the rationale to examine CLA’s therapeutic potential in the context of a Candida infection. There is a general lack of clinical evidence demonstrating that impairing morphogenesis is a sound approach to treat candidiasis. To remedy this situation, the therapeutic potential of molecules that modulate morphogenesis, such as CLA, should be clinically assessed.
694

Peroxidases regulation of Candida albicans oral biofilms / Effet régulateur des peroxydases sur les biofilms oraux à Candida albicans

Ahariz, Mohamed 15 March 2012 (has links)
Among the prosthetic and implant biomaterials in the oral cavity currently exciting tremendous interest, titanium and resin are the main components of implants and dentures respectively. From their introduction in the oral cavity, a highly complex heterogeneous biofilm coats these biomaterials. The present thesis analyzes the complex relationships that are formed between one single microorganism (Candida albicans), one defence system of the oral cavity (oral peroxidases) and the two aforementioned biomaterials (titanium and resin). These biomaterials are indeed in contact with peroxidases: myeloperoxidase from neutrophils and sialoperoxidase from salivary secretions. Oral peroxidases belong to the salivary non immune innate defence mechanisms that control the oral microbial flora. In the presence of hydrogen peroxide (H2O2), they catalyze in vivo the oxidation of thiocyanate (SCN-) into hypothiocyanite (OSCN-), in vitro the oxidation of iodide (I-) into hypoiodite (OI-). In the salivary compartment and in oral biofilms, H2O2 is mainly formed by bacteria. In our investigations, H2O2 was produced by an enzyme sequence glucose (G) / glucose-oxidase (GOD). The OSCN- and OI- are antibacterial, antiviral and antifungal oxidants. Few studies have considered their action on Candida biofilms.<p>Candida albicans is a commensal yeast of the oral cavity which can turn parasitic when the host immune defences are weakened. This fungus forms biofilms on biomaterials within the mouth, especially on dentures, the decontamination of these prostheses is therefore essential to avoid the risk of candidosis. (Candida et prothèses dentaires. Ahariz M. Loeb I. Courtois Ph. Rev. Stomatol. Chir. Maxillofac. 111: 74-78, 2010).<p>In vitro, our investigations aimed to analyze the relationships between peroxidase systems and Candida. The effect of peroxidase systems (G / GOD / KI or KSCN / peroxidase) on Candida suspensions, on biofilms already formed or in formation was evaluated with various inputs of hydrogen peroxide and was studied by incorporation of enzymatic sequences in the culture media used for Candida biofilms formation. The susceptibility of Candida albicans ATCC 10231 to OSCN- versus to OI-, produced by lactoperoxidase (LPO), was studied in three different experimental models: - in a liquid culture medium – on a solid medium (with agarose gel), - in a biofilm model developed in the context of this work. The latter consisted of titanium powder suspended in Sabouraud broth contaminated with Candida albicans. The growth of Candida in the supernatant (planktonic phase) was evaluated by turbidimetry and the biomass of yeasts adherent to biomaterials (attached phase) by the tetrazolium salt MTT method. Enzymatic studies have allowed the optimization of the concentrations and activities of peroxidase systems components and the illustration of the competition between thiocyanate and iodide for lactoperoxidase. Peroxidase systems G/GOD/I-/LPO and G/GOD/SCN-/LPO prevented or limited the growth of Candida in the planktonic and attached phases on titanium powder for at least 21 days. At a dose of GOD (0.2 U / ml), the system G/GOD/I-/LPO has limited the development of planktonic and attached phases for 4 days while the system G/ GOD/SCN-/LPO has shown an inhibitory effect only in the first 2 days of incubation. (Candida albicans susceptibility to lactoperoxidase-generated hypoiodite. Ahariz M. Courtois Ph. Clinical, cosmetic and investigational dentistry; 2: 69-78, 2010).<p>In other experiments, peroxidase was adsorbed on titanium sheets in order to modify their surface and give them the property of inhibiting biofilm formation of Candida after addition of the enzyme substrates. Enzymatic studies and X-ray photoelectron spectroscopy (XPS) showed the adsorption of lactoperoxidase to titanium. In vivo, peroxidases are adsorbed on titanium healing abutments. (Adsorption of peroxidase on titanium surfaces: A pilot study. Ahariz M. Mouhyi J. Louette P. Van Reck J. Malevez C. Courtois P. J. Biomed. Mater. Res. 52: 567-571, 2000).<p>The development of the Candida biofilm was followed on titanium (powder or sheets) as well as on resin. Planktonic and attached phases have been monitored for 21 days. The presence of an exopolysaccharide matrix secreted by the yeasts has been observed with light microscopy and confirmed with fluorescence using the calcofluor method. In a series of experiments on titanium and resin sheets, an attached phase was demonstrated by the same techniques. The efficiency of the peroxidase system using iodide as a substrate was demonstrated when the enzyme was in solution and when it was preadsorbed on titanium (Candida albicans biofilm on titanium: effect of peroxidase precoating. Ahariz M. Courtois Ph. Medical Devices: Evidence and Research; 3: 33-40, 2010).<p>The incorporation in oral gels of other molecules present in exocrine secretions is a research direction that was also discussed: the present studies have demonstrated the in vitro benefits of peroxidase systems (with thiocyanate, chloride or especially iodide as substrates) acting in synergy with colostrum, lactoferrin and lysozyme. But the formulation of specialities that contain these natural antimicrobials is difficult to transpose in vivo as the complexity of the oral environment is very large (Denture contamination by yeasts in the elderly. Vanden Abbeele A. de Meel H. Ahariz M. Perraudin J.-P. Beyer I. Courtois P. Gerodontology; 25: 222-228, 2008).<p>Investigations pursued in vivo in 155 patients allowed the determination of the wild strains of Candida sp present on the fitting surface of the removable dental appliance and on the corresponding palatal mucosa. The link between the presence of yeasts and a reduced salivary flow was confirmed. These wild strains were directly grown and identified on Petri dishes (ChromAgar™ medium) from the macroscopic morphology of colonies and from additional tests (germination test in human serum, formation of chlamydoconidies on RAT medium, API™ galleries identification system ). For a period of two weeks, 14 patients accepted the daily application of a gel on the fitting surface of their denture. It was a double-blind comparison of an active gel containing the thiocyanate - lysozyme - lactoferrin - colostrum complete system with a control gel inactivated by heating. Data analysis showed a reduction in the number of colonies on the palatal mucosa by Candida sp, but not on the denture itself. By contrast, decontamination ex vivo of dentures by immersion in a bath (at room temperature or 37° C) containing either G/GOD producing H2O2 or the complete peroxidase system G/GOD/KI/L producing OI- demonstrated the efficiency of hypoiodite.<p><p><p>Parmi les biomatériaux prothétiques et implantaires qui connaissent actuellement un essor considérable dans la sphère orale, le titane est le composant principal des implants et la résine celui des prothèses dentaires. Dès leur introduction dans la cavité orale, un biofilm hétérogène très complexe les recouvre. Cette thèse analyse la complexité des relations qui se nouent entre un seul micro-organisme (Candida albicans), un système de défense de la cavité orale (les peroxydases orales) et les 2 biomatériaux précités (titane, résine). Ces biomatériaux sont en effet, dans le milieu oral, au contact de peroxydases: la myéloperoxydase des neutrophiles et la sialoperoxydase des sécrétions salivaires. Les peroxydases orales appartiennent aux mécanismes salivaires de défense innée non immunitaires qui contrôlent la flore microbienne orale. En présence de peroxyde d’hydrogène (H2O2), elles catalysent in vivo l’oxydation du thiocyanate (SCN-) en hypothiocyanite (OSCN-) et in vitro l’oxydation d’iodure (I-) en hypoiodite (OI-). Dans le compartiment salivaire et dans les biofilms oraux, l’H2O2 provient essentiellement des bactéries. Dans nos investigations, l’H2O2 était produit par une séquence enzymatique glucose (G) / glucose-oxydase (GOD). L’OSCN- et l’OI- sont des oxydants antibactériens, antiviraux et antifongiques. Peu d’études envisagent leur action sur les biofilms à Candida.<p>\ / Doctorat en Sciences dentaires / info:eu-repo/semantics/nonPublished
695

Isolierung, Identifizierung und funktionelle Charakterisierung der Metallo-Aminopeptidase CaApe2 — Ein experimenteller Beitrag zur Beurteilung des kariogenen Potentials von Candida albicans

Pönisch, Roman 09 December 2008 (has links)
Die Hefe Candida albicans ist ein fakultativ humanpathogener Mikroorganismus, der insbesondere bei immungeschwächten Patienten schwere Erkrankungen der Haut und Schleimhäute sowie der inneren Organe hervorrufen kann. Seit langer Zeit wird eine Beteiligung des Hefepilzes an der Ätiopathogenese der Zahnkaries diskutiert, vor allem aufgrund der Säure-bildung, die zur Demineralisation der Zahnhartsubstanz beitragen kann. Hydrolytische Enzyme ermöglichen vermutlich die Gewebeinvasion von Candida albicans. In der vorliegenden Arbeit wurde ein sezerniertes peptidolytisches Enzym aus der Zellwand des Mikroorganismus isoliert, identifiziert und funktionell charakterisiert. Die mittels massenspektrometrischer Analyse der tryptischen Peptide und Datenbankrecherche ermittelte Primärstruktur und die Ergebnisse der funktionellen Charakterisierung ließen eine Identifi-zierung des peptidolytischen Enzyms als neutrale Arginin/Alanin/Leucin-spaltende Metallo-Aminopeptidase (CaApe2) zu, die durch den ORF CaO19.5197 (GenBank RefSeq XM 705313) kodiert wird. Mithilfe der Proteinanalytik wurde Serin-88 als N-terminale Aminosäure ermittelt. Die Aminosäuren 88 bis 954 des hypothetischen Genprodukts ergeben eine nominale Molekularmasse von 97,607 kDa. CaApe2 weist gleich hohe Ähnlichkeit mit den paralogen Genprodukten ScAap1 und ScApe2 auf, was eine Duplikation und Subfunktionalisierung des phylogenetischen Vorläufergens in Saccharomyces cerevisiae nahe legt. Die fehlende kollagenolytische Wirksamkeit von CaApe2 spricht gegen eine direkte Rolle des Enzyms in der Pathogenese der Dentinkaries von Candida albicans, schließt aber eine unterstützende Funktion nicht aus. Die Kollagendegradation durch aufgeschlossene Zellen und Kulturüberstand einer Flüssigkultur von Candida albicans wurde im sauren und neutralen Milieu mithilfe der Hydroxyprolin-Bestimmung untersucht. Dabei war keine Kollagenolyse mit Aktivitätsmaximum im neutralen Bereich nachweisbar. Im sauren pH-Bereich konnte eine deutliche Hydrolyse von säureunlöslichem Typ-I-Kollagen und auch von demineralisierter Dentinmatrix durch Kulturmedium gezeigt werden. Diese Kollagenolyse kann auf die bereits umfangreich charakterisierten sezernierten Aspartylproteinasen zurückgeführt werden. Die in der Literatur beschriebene Korrelation zwischen dem Ausmaß des Kariesbefalls und der Quantität der Besiedelung mit Candida albicans legt eine Beteiligung des Hefepilzes an der Kariogenese nahe. Auch die in der vorliegenden Arbeit gezeigte Fähigkeit von Candida albicans zur Dentinkollagendegradation unterstützt die Hypothese einer Kariogenität der Hefe. / The proteolytic potential of the pathogenic fungus Candida albicans was evaluated by the identification and functional characterization of a peptidolytic enzyme isolated from the cell wall of the microorganism. Determination of basic structural and kinetic data identified a neutral arginine/alanine/leucine-specific metallo-aminopeptidase of unknown function termed CaApe2 which is encoded by ORF CaO19.5197 (GenBank RefSeq XM_705313). Mass spectrometric tryptic peptide analysis and N-terminal protein sequencing revealed serine-88 to represent the N-terminus of CaApe2. The isolated CaApe2 protein shares equally high similarity with the gene products ScAap1 and ScApe2 suggesting duplication of a phylogenetically ancient precursor gene in Saccharomyces cerevisiae. The observed failure to cleave human type-I and type-IV collagen in vitro challenges a direct role secreted CaApe2 might play in the degradation of extracellular matrix components during host colonization, but does not exclude per se a contribution of the aminopeptidase to the pathogenicity of C. albicans.
696

Interaction Studies of Secreted Aspartic Proteases (Saps) from Candida albicans : Application for Drug Discovery

Backman, Dan January 2005 (has links)
This thesis is focused on enzymatic studies of the secreted aspartic proteases (Saps) from Candida albicans as a tool for discovery of anti-candida drugs. C. albicans causes infections in a number of different locations, which differ widely in the protein substrates available and pH. Since C. albicans needs Saps during virulent growth, these enzymes are good targets for drug development. In order to investigate the catalytic characteristics of Saps and their inhibitor affinities, substrate-based kinetic assays were developed. Due to the low sensitivity of these assays, especially at the sub-optimal pH required to mimic the different locations of infections, these assays were not satisfactory. Therefore, a biosensor assay was developed whereby, it was possible to study interaction between Saps and inhibitors without the need to optimise catalytic efficacy. Furthermore, the biosensor assay allowed determination of affinity, as well as the individual association and dissociation rates for inhibitor interactions. Knowledge about substrate specificity, Sap subsite adaptivity, and the pH dependencies of catalytic efficacy has been accumulated. Also, screening of transition-state analogue inhibitors designed for HIV-1 protease has revealed inhibitors with affinity for Saps. Furthermore, the kinetics and pH dependencies of their interaction with Saps have been investigated. One of these inhibitors, BEA-440, displayed a complex interaction with Saps, indicating a conformational change upon binding and a very slow dissociation rate. A time dependent interaction was further supported by inhibition measurements. The structural information obtained affords possibilities for design of new more potent inhibitors that might ultimately become drugs against candidiasis. The strategy to combine substrate specificity studies with inhibitor screening has led to complementary results that generate a framework for further development of potent inhibitors.
697

Caractérisation génétique, phénotypique et formation de biofilm des souches de Candida albicans répondant ou non au farnésol

Irimes, Cristina 12 1900 (has links)
Candida albicans, le pathogène opportuniste le plus commun, peut subir des transitions morphologiques entre la forme levure et la forme hyphe, jouant un rôle dans la formation de biofilm. Le farnésol, un lipide endogène produit par C. albicans, est une molécule de quorum sensing qui inhibe cette transition morphologique. Certaines souches ne répondent pas au farnésol et nous avons vérifié les hypothèses que : 1) l’isolat clinique SC5314, la souche la mieux caractérisée, est un répondeur au farnésol; 2) la germination, la croissance et la formation de biofilm des non répondeurs diffèrent des répondeurs; 3) l’absence de la réponse au farnésol se manifeste en dehors de conditions de culture précises; 4) le farnésol agit via un récepteur nucléaire qui présente des altérations chez les non répondeurs; 5) la différence de la réponse au farnésol entre les souches s’explique par des variations au niveau transcriptionnel de certains gènes (CHK1, HST7, CPH1, GAP1, RAM2 et DPP3). Les non répondeurs produisent un plus grand nombre d’hyphes, forment 60% plus de biofilm et croissent 50% moins vite que les répondeurs. La souche SC5314 se comporte comme un répondeur. L’absence de la réponse au farnésol se manifeste indépendamment des conditions de culture. Cependant, elle ne s’explique pas par une différence dans le niveau d’expression des gènes proposés, excepté pour DPP3 qui est surexprimé chez le non répondeur ATTC® 36802, suggérant ainsi une surproduction de farnésol chez cette souche. De plus, si le farnésol agit via un récepteur nucléaire, il sera d’un type non décrit précédemment. / Candida albicans, the most common fungal pathogen, can undergo morphological transitions between yeast and hyphal forms, which are associated with biofilm formation. Farnesol, an endogenous lipid produced by C. albicans, is a quorum sensing molecule that inhibits this transition. Previous work identified two clinical isolates that didn’t respond to farnesol in colony morphology and biofilm assays. Our goal is to better understand C. albicans response to farnesol using these natural farnesol non responders. We have hypothesized that : 1) clinical isolate SC5314, the most characterized strain, is a farnesol responder; 2) non responders’ germination, growth and biofilm formation are different from those of responders; 3) lack of response to farnesol occurs even outside specific culture conditions; 4) farnesol acts through a nuclear receptor that is altered in non responders; 5) difference in farnesol response between strains is explained by transcriptional variations of specific genes (CHK1, HST7, CPH1, GAP1, RAM2 and DPP3), that were previously shown to be potentially involved in farnesol’s mechanism of action. Non responders produce more hyphae, form 60 % more biofilm and grow 50% slower than responders. The SC5314 strain acts like a responder. Lack of response to farnesol occurs regardless of culture conditions. However, the refractory response to farnesol is not explained by a difference in the proposed genes expression level, except for DPP3 that is upregulated in ATTC® 36802 non responder, suggesting an overproduction of farnesol by this strain. Furthermore, if farnesol acts trough a nuclear receptor, it will be a type not previously described.
698

Cell Wall/Surface Proteome of Candida albicans: its Application in Rapid Identification of Yeast Species by Mass Signature and Characterization by in vitro and in vivo Chemical Labelings

Qian, Jiang 14 May 2010 (has links)
Candida albicans is an opportunistic fungal pathogen that may cause mucutaneous infection and/or disseminated candidasis if the host defense system is impaired (such as those in HIV patients). Cell surface of C. albicans is the frontier where initial interplay between host-pathogen takes place and therefore is of great importance in understanding the mechanism of hostpathogen interaction. MALDI-TOF-MS analysis of intact fungal cells yielded mass signatures for rapid species differentiation, strain grouping and yeast morphogenesis monitoring. Cell surface biotinylations at low temperature (4°C), enzymatic digestion of the intact fungal cell surface proteins ("whole cell shaving"), biotin-avidin affinity enrichment of biotinylated peptides, liquid chromatography mass spectrometry (LC-MS) based proteomic approach were employed for unambiguous identification of cell wall/cell wall associated proteins and the exposed peptide segments of these proteins. SILAC (Stable Isotope Labeling by Amino acids in Cell Culture) based CWP quantification analyses were performed to monitor CWP accumulation level change in response to hyphae induction. Information on surface exposed peptide segments and regulation of cell wall/surface protein during morphogenesis provided new candidates to the pool of potential peptide targets for protective vaccine development. A New type of "fluorous" (fluorinated alkane) affinity gained popularity due to its low level nonspecific protein/peptide binding. Fluorous labeling reagents that target primary amine groups in proteins/peptides were synthesized and characterized. The acid labile linker in the labeling reagents allows cleavage of the bulky fluorous tag moiety and the long oligo ethylene glycol (OEG) spacer after fluorous affinity purification. Upon collision induced decomposition, the labeled peptide ion yielded a characteristic fragment that could be retrieved from the residual portion of fluorous affinity tag, and serve as a marker to indicate that the relevant peptide had been successfully labeled. Results showed that both the protein/peptide labeling and affinity enrichment/separation process were highly efficient.
699

O efeito do uso de fitoterápicos e da própolis nas propriedades físico-químicas, antimicrobiana e biocompatibilidade do MTA / The effect of the use of phytotherapics and propolis in physicochemical properties, antimicrobial and biocompatibility of MTA

Cavenago, Bruno Cavalini 31 March 2015 (has links)
O objetivo deste estudo foi avaliar as propriedades físico-químicas, antimicrobianas e biocompatibilidade do MTA branco manipulado com extratos aquoso e/ou em propilenoglicol da Arctium lappa L., Casearia sylvestris Sw. e própolis. Dentre os testes físico-químicos foram avaliados o tempo de presa, escoamento, pH, liberação de íons cálcio e alteração volumétrica. Para verificar o efeito antimicrobiano foram aplicadas as metodologias do contato direto (Enterococcus faecalis e a Cândida albicans) e da descontaminação dentinária, empregando a microscopia confocal de varredura laser para verificar a viabilidade de Enterococcus faecalis. Para a avaliação da biocompatibilidade, 162 ratos Wistar foram utilizados, onde cada animal recebeu dois implantes subcutâneos e um alveolar. Após os períodos experimentais de 15, 30 e 60 dias foram realizadas análises microtomográfica, histológica descritiva e histomorfométrica. Adicionalmente amostras do tecido alveolar foram processadas para dosagem das citocinas TNF-&#x3B1; e IL-10 por meio do ensaio imunoenzimático (ELISA). Os dados obtidos foram analisados estatisticamente com os testes ANOVA e Tukey ou Kruskal-Wallis e Dunn. Os resultados revelaram que a variação do veículo associado ao MTA aumentou significativamente o tempo de presa, no entanto, não houve influência na alteração volumétrica (P>0,05) e na capacidade do cimento em manter o meio alcalino e liberar íons cálcio. Os cimentos manipulados com extratos em propilenoglicol apresentaram maior escoamento (P<0,05). Apenas o extrato da própolis agregou ao MTA efeito contra o Enterococcus faecalis após 24 e 48 horas (descontaminação dentinária e contato direto respectivamente) e contra a Cândida albicans após 10 horas (P<0,05). De acordo com as avaliações histológica e histomorfométrica dos implantes em tecidos subcutâneo e alveolar não foi constatada diferença significativa entre os grupos experimentais quando comparados com o grupo no qual o MTA foi manipulado com água destilada. Segundo a análise microtomográfica e a expressão das citocinas TNF-&#x3B1; e IL-10 houve similaridade (P>0,05) entre todos os grupos. Apesar de ter alterado algumas propriedades físico-químicas, o extrato da própolis potencializou o efeito antimicrobiano do MTA sem, contudo, alterar sua biocompatibilidade. / The aim of this study was to evaluate the physicochemical, antimicrobial properties and biocompatibility of white MTA mixed with aqueous or propylene glycol extracts of Arctium lappa L., Casearia sylvestris Sw. and propolis. Among physicochemical tests were evaluated the setting time, flowability, pH, ion calcium release and volumetric change. To verify the antimicrobial effects were applied the methods of direct contact (Enterococcus faecalis and Candida albicans) and dentin decontamination by using the confocal laser scanning microscopy to verify the Enterococcus faecalis viability. To evaluate the biocompatibility were used 162 Wistar rats. Each animal received one alveolar and two subcutaneous implants. After the experimental periods of 15, 30 and 60 days were performed the microtomography, histological description and histomorphometric analyses. Additionally alveolar tissue samples were processed for the measurement of TNF-&#x3B1; e IL-10 cytokines by enzyme-linked immunosorbent assay (ELISA). The data were statistically analyzed by the ANOVA and Tukey or KruskalWallis and Dunns tests. The results revealed that the variation of the vehicle associated to MTA significantly increased its setting time, however did not influence the volumetric change (P>0,05) and the cement\'s ability to maintain the alkaline medium and ion calcium release. Cements mixed with propylene glycol extracts showed higher flowability (P<0,05). Only propolis extract added to MTA the effect against E. faecalis after 24 and 48 hours (dentin decontamination and direct contact respectively) and against Candida albicans after 10 hours (P<0,05). According to the histological and histomorphometric evaluation of the implants in subcutaneous and alveolar tissue was not observed significant differences between the experimental groups in comparison to the reference group (MTA was mixed with distilled water). The microtomography analysis and expression of TNF-&#x3B1; and IL-10 showed that the groups were similar (P>0,05). Although the propolis extract modified some physicochemical properties of MTA it is potentiated the antimicrobial effect and did not influence the biocompatibility.
700

Avaliação in vitro da atividade antifúngica de óleos essenciais e extratos de plantas presentes na região amazônica sobre cepa de Candida albicans

MENEZES, Tatiany Oliveira de Alencar January 2008 (has links)
Submitted by Cleide Dantas (cleidedantas@ufpa.br) on 2014-02-14T16:35:42Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_AvaliacaoInvitroAtividade.pdf: 2004391 bytes, checksum: 2ffa3100ba85604a60ae23cddad1145f (MD5) / Approved for entry into archive by Ana Rosa Silva (arosa@ufpa.br) on 2014-04-30T15:37:31Z (GMT) No. of bitstreams: 2 Dissertacao_AvaliacaoInvitroAtividade.pdf: 2004391 bytes, checksum: 2ffa3100ba85604a60ae23cddad1145f (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) / Made available in DSpace on 2014-04-30T15:37:32Z (GMT). No. of bitstreams: 2 Dissertacao_AvaliacaoInvitroAtividade.pdf: 2004391 bytes, checksum: 2ffa3100ba85604a60ae23cddad1145f (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Previous issue date: 2008 / A candidíase é uma doença fúngica oportunista causada pela proliferação de espécies de Candida, principalmente a Candida albicans, sendo a espécie mais patogênica em humanos. Muitos antifúngicos existentes no mercado apresentam efeitos colaterais indesejáveis ou podem induzir a resistência fúngica, principalmente em indivíduos imunodeprimidos. Em odontologia, as pesquisas com produtos naturais têm aumentado nos últimos anos, devido à busca por novos produtos com maior atividade farmacológica, com menor toxicidade e mais acessíveis à população. Dentro dessa perspectiva, o objetivo desta pesquisa foi avaliar in vitro a atividade antifúngica de óleos e extratos vegetais presentes na região Amazônica e determinar a concentração inibitória mínima das espécies que apresentaram atividade antifúngica frente à cepa padrão de Candida albicans (ATCC 90028). A atividade antifúngica dos óleos essenciais Copaifera multijuga, Carapa guianenses, Piper aduncum e Piper hispidinervum foi realizada pelo método de difusão em meio sólido utilizando cavidades em placa “in natura” e em diluições de 32 a 2% para determinação da concentração inibitória mínima. Os extratos Annona glabra, Azadiractha indica, Bryophyllum calycinum, Eleutherine plicata, Mammea americana, Psidium guajava e Syzygium aromaticum foram testados nas concentrações de 500mg/mL, 250mg/mL, 125mg/mL e 62,5 mg/mL e a atividade antifúngica foi realizada pelo método de difusão em meio sólido utilizando discos de papel filtro. Os óleos testados, não apresentaram efeito antifúngico sobre a cepa de Candida albicans, e dos extratos testados somente os extratos de Eleutherine plicata, Psidium guajava e Syzygium aromaticum apresentaram atividade antifúngica com concentração inibitória mínima, respectivamente, de 250mg/mL, 125mg/mL e 62,5mg/mL Diante dos resultados apresentados, os extratos de Eleutherine plicata, Psidium guajava e Syzygium aromaticum apresentam potencial efeito inibitório para crescimento de Candida albicans, servindo de guia para a seleção de plantas com atividades antifúngicas para futuros trabalhos toxicológico e farmacológico. / Candidiasis is an opportunistic fungus disease caused by the proliferation of Candida species, mainly Candida albicans, what seems to be the most pathogenic one to humans. Several anti-fungii drugs available in the market present undesirable side effects or develop resistance, mainly in immune-depressed individuals. In dentistry, researches with natural products have increased in the last years due to the search for new products with a greater pharmacologic activity, lower toxicity and more accessibility to the population. Hence, the aim of this paper was to evaluate in vitro the anti-fungii activity of vegetal oils and extracts present in the Amazon region and to determine the minimal inhibitory concentration of such species that have presented antifungii activity against the pattern strain of Candida albicans (ATCC 90028). The antifungii activity of the essential oils Copaifera multijuga, Carapa guianenses, Piper aduncum and Piper hispidinervum was determined through the method of difusion in a solid means using the cavities in plaque “in natura” and in dilutions of 32 to 2% in order to determine its minimal inhibitory concentration. The extracts Annona glabra, Azadiractha indica, Bryophyllum calycinum, Eleutherine plicata, Mammea americana, Psidium guajava and Syzygium aromaticum have been tested under the concentrations of 500mg/mL, 250mg/mL, 125mg/mL and 62,5 mg/mL and the anti-fungii activity was determined through the method of difusion in a solid means using discs of filter paper. The tested oils did not present any anti-fungii effect on the strain of Candida albicans and, from the extracts tested, only Eleutherine plicata, Psidium guajava and Syzygium aromaticum have presented anti-fungii activity with minimal inhibitory concentrations, respectively, 250mg/mL, 125mg/mL and 62,5mg/mL With the results presented, the extracts of Eleutherine plicata, Psidium guajava and Syzygium aromaticum present a potential inhibitory effect to the growth of Candida albicans, acting as a guide for the selection of plants with anti-fungii activities for further papers on a toxicologic and pharmacologic.

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