O antisséptico cloreto de cetilpiridíneo (CCP) demonstrou ser eficaz como alternativa terapêutica aos antifúngicos em candidose oral, porém, apresenta baixa substantividade. Para aumentar sua biodisponibilidade surge a possibilidade de incorporá-lo num sistema de liberação lenta composto por filmes poliméricos nanofibrílicos. O objetivo deste estudo foi preparar filmes nanofibrílicos através de eletrofiação e incorporar CCP, fazer sua caracterização térmica e morfológica, além de análise microbiológica dos filmes contendo CCP frente a cepas de Candida albicans. O fármaco CCP foi adicionado a três soluções poliméricas, de álcool polivinílico (PVA), polivinilpirrolidona (PVP) e a de PVP/PM [Poli (metacrilato de metila-co-acrilato de etila-co-metacrilato de amônio)] em 5% de concentração. As soluções foram avaliadas quanto à condutividade, viscosidade e tensão superficial e, posteriormente, passaram pelo processo de eletrofiação para obtenção de filmes nanofibrílicos. Os filmes nanofibrílicos foram caracterizados através de análise morfológica (microscopia eletrônica de varredura - MEV), análise térmica (análise termogravimétrica - TGA e calorimetria exploratória diferencial - DSC) e análise química (espectro de infravermelho da transformada de Fourier - FTIR). A eficiência de encapsulação de CCP nas nanofibras, o perfil cinético de liberação das nanofibras e a permeação em mucosa suína (células de Franz) foram avaliados por espectrofotometria. Foi ainda avaliada a concentração de CCP a ser incorporada (de 0,05 a 5%) em nanofibra para que a fração liberada seja a mínima fungicida frente às cepas de C. albicans susceptíveis, pela técnica de disco-difusão, comparada ao miconazol 5% (MCZ). Nestas condições, a solução de PVA apresentou a maior condutividade, viscosidade e tensão superficial. CCP aumentou a condutividade. Ambos, CCP e PM não alteram a tensão superficial do PVP. PM reduziu a viscosidade. A solução de PVP/PM formou o filme mais uniforme e com menos beads. Os termogramas (TGA/DSC) sugerem que o processo de eletrofiação diminiu a cristalinidade de CCP. A eficiência de encapsulação de CCP alcança 99%. A taxa de liberação de CCP encapsulado foi lenta durante o período experimental de 24h. Quando em filme nanofibrílicos, a atividade fungicida de CCP 2,5% e de MCZ 5% foi similar, mas somente MCZ foi fungistático. O filme nanofibrílico contrai em contato com o meio de cultura. Pode-se concluir que filmes nanofibrílicos de PVP/PM com CCP tem potencial para uso como alternativa para tratamento de infecções orais causadas por Candida. / The aim of this study was to prepare electrospun nanofiber films cetylpyridinium chloride (CPC) incorporated test their potential use as antifungal therapy against Candida albicans. CPC was incorporated to three different polymeric solutions, one containing polyvinyl alcohol (PVA), the second containing polyvinylpyrrolidone (PVP) and the third prepared with PVP and poly (methyl methacrylate-co-ethyl acrylate-co-methacrylate ammonium) (PMMA). These polymeric solutions were evaluated as to conductivity, viscosity and surface tension, then subjected to a electrospinning process to obtain nanofiber films. The morphology and structure of nanofiber films were analyzed using scanning electron microscopy (SEM). The characterization of nanofiber films was performed by thermogravimetric analysis (TG) and differential scanning calorimetry (DSC). The encapsulation efficiency of the CPC nanofibers and the release kinetic profile of CPC and porcine mucosa permeation (Franz cells) were assessed by spectrophotometry. It was also assessed the concentration of CPC to be incorporated (from 0.05 to 5%) in nanofiber to be minimal fungicidal fraction released against strains of C. albicans by disk diffusion tests, compared to 5% miconazole (MCZ). Under these conditions, the PVA solution showed the highest conductivity, viscosity and surface tension. CPC increased conductivity. Both CPC and PMMA did not alter the surface tension of PVP. PMMA reduced viscosity. The solution of PVP/PMMA formed a more uniform film with less beads. Thermograms (TGA / DSC) suggested that the electrospinning process changes the crystallinity of CPC. The encapsulation efficiency reaches 99% of CPC. The release rate of encapsulated CPC was slow during the experimental period of 24 hours. In a nanofibrilic film, the fungicidal activity of CPC 2.5% and 5% MCZ was similar, but only MCZ proved fungistatic action. The nanofibrilic film shows contraction in contact with the culture medium. It can be concluded that nanofibrilic films PVP/PMMA with CPC has potential for use as an alternative for the treatment of oral infections caused by Candida.

Uma infecção pulpar pode resultar na colonização microbiana de todo sistema de canais radiculares incluindo os túbulos dentinários. Estes microorganismos e seus produtos tóxicos são responsáveis pelo desenvolvimento e persistência da periodontite apical de origem endodôntica. O presente estudo objetivou avaliar a viabilidade de E. faecalis e C. albicans em túbulos dentinários após a aplicação de hidróxido de cálcio, clorexidina gel 2%, hidróxido de cálcio associado à clorexidina gel 2% e soro fisiológico, através da análise por cultura microbiológica e microscopia de fluorescência. Para tanto 120 raízes de dentes humanos foram padronizadas e autoclavadas, sendo posteriormente divididas em 2 grupos (n= 60) para contaminação com E. faecalis e C. albicans por 21 dias. Em seguida, foram divididas em 8 grupos (n= 15) para aplicação das substâncias antimicrobianas nos canais radiculares e posterior incubação em estufa por 14 dias. Amostras da dentina radicular na extensão de 0 - 100 µm e de 100 - 200 µm foram coletadas e submetidas à cultura microbiológica através do plaqueamento em meios de cultura. Após 48 horas de incubação promoveu-se a avaliação das UFC. Paralelamente, as amostras foram processadas para análise em microscopia de fluorescência com auxílio de marcadores fluorescentes específicos, a fim de se determinar a proporção de microorganismos viáveis e não viáveis. Outros 6 espécimes foram preparados para análise em MEV. Os resultados mostraram uma maior capacidade de penetração intratubular para E. faecalis quando comparado a C. albicans. A aplicação de medicação intracanal resultou em significativa redução da viabilidade dos microorganismos quando comparado ao grupo controle independente da medicação aplicada e em ambas as porções da dentina radicular avaliadas. Entretanto, ao compararmos individualmente as medicações, observamos o melhor desempenho da clorexidina gel 2 % e da associação de hidróxido de cálcio e clorexidina gel 2% sem diferença significante entre elas. O hidróxido de cálcio apresentou os piores resultados para desinfecção dos canais radiculares contaminados com E. faecalis e C. albicans. Estes achados foram confirmados tanto pela cultura microbiológica quanto pela microscopia de fluorescência. A cultura microbiológica e a microscopia de fluorescência são métodos adequados e complementares para avaliação da viabilidade de E. faecalis e C. albicans e a eficácia da clorexidina gel 2% e da associação hidróxido de cálcio e clorexidina gel 2% justificam seu uso em endodontia como medicação intracanal. / A pulp infection can result in a microbial colonization of the entire root canals system, including dentinal tubules. These microorganisms and their toxic product are responsible for the development and persistence of apical periondontitis from endodontic source. The present study aimed to evaluate E. faecalis and C. albicans viability in dentinal tubules after the application of calcium hydroxide, chlorhexidine gel 2%, calcium hydroxide associated to chlorhexidine gel 2% and physiological solution, through the analysis by microbiological culture and fluorescence microscopy. For that, 120 human teeth root were standardized and submitted to autoclave, and after ere divided into 2 groups (n=60) for E. faecalis and C. albicans contamination for 21 days. Following this, they were divided into 8 groups (n=15) for application of antimicrobial substances in the root canals and subsequent incubation for 14 days. Samples from root dentin with 0 - 100 µm and 100 - 200 µm of extension were collected and submitted to microbiological culture.After 48 hours of incubation, it was performed the evaluation of colony-forming units (CFU). At the same time, the samples were processed for fluorescence microscopic analysis with the assistance of specific fluorescent markers, in order to determine the proportion of viable and non-viable microorganisms. Other 6 specimens were prepared for the analysis of scanning electron microscopy. Results demonstrated a higher capacity of penetration in the tubules for E. faecalis than for C. albicans. The application of intracanal medication resulted in significant reduction of microorganisms viability when compared to the control groups independently of the medication used and in both evaluated portions of root dentin. However, when one compares individually the medications, it was observed a better performance of chlorhexidine gel 2% and the association of calcium hydroxide with chlorhexidine gel 2% without a significant difference between them. Calcium hydroxide had the worst results for disinfection of root canal contaminated with E. faecalis and C. albicans. These findings were confirmed for the microbiological culture as well as for the fluorescence microscopy. The microbiological culture and the fluorescence microscopy are adequate methods and complementary for the evaluation of E. faecalis and C. albicans viability and the efficacy of chlorhexidine gel 2% and the association of calcium hydroxide with chlorhexidine gel 2% warrant their use in Endodontics as an intracanal medication.

Made available in DSpace on 2017-07-24T19:21:59Z (GMT). No. of bitstreams: 1 Andres Felipe.pdf: 3893759 bytes, checksum: fd56d743b750b678334823fb1406f10a (MD5) Previous issue date: 2016-02-05 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The combination of well-fitting dentures with topical antifungals is appropriate therapeutic approach for denture stomatitis (EP). It was developed and evaluated an adhesive for removable dentures containing miconazole nitrate (NM) incorporated into mucoadhesive and/or pH dependent polymer microparticles aiming at increasing bioavailability. Initially, microparticles have been developed containing 10% and 20% of NM, spray-drying, using Gantrez MS-955 polymer (G10, G20), Eudragit L-100 (E10, E20) or both (EG10, EG20). An analytical method by high-performance liquid chromatography (HPLC) to quantify NM of the microparticles was validated. Microparticles were characterized by scanning electron microscopy (SEM), x-ray diffraction, Fourier-transformed infrared spectrometry (FTIR), differential scanning calorimetry (DSC), in-vitro release studies (percentage of dissolution / time, and release profiles) and antifungal activity. An experimental denture adhesive formulation (ACT) was developed containing 10% by weight of the microparticles (AE1, AG1, AEG1, AE2, AG2, AEG2) or 2% of pure drug (ANM). For all adhesives it was determined: minimum inhibitory concentration (MIC) for Candida albicans (microdilution and agar dilution); adhesive force (FA among acrylic surfaces after 0.5, 1, 3, or 6 h immersion in water); and toxicity to brine shrimp (24 h and 48 h) by calculating lethal concentration 50 (LC50). The HPLC method was proven specific, linear (r = 0.9992), precise, accurate and robust in the range 5-90 μg.mL-1, with running and retention times of 10.0 and 5.58 minutes, respectively. All microparticles showed acceptable performance (37.22% - 55.36%) and encapsulation (over 89%) values. E10 and E20 microparticles showed spherical and smooth surface, while EG20 had similar shape, but rough surface. G10, G20 and EG20 had depressed craters and morphology. The diameters of the microparticles ranged from 1.9 to 4.3 micrometers. No chemical bond was observed between the MN and the polymers through the FTIR spectra. Microencapsulation contributed to the drug amorphization, according to thermal analysis and X-ray diffraction, reducing the release time. NM and the G10, G20 and EG20 microparticles fitted the biexponential release kinetic model and the microparticles E10, E20 and EG10 fitted to mono-exponential model. The microparticles showed antifungal efficiency similar to pure drug. Extracts of the adhesives containing the microparticles and ANM showed MIC of 1.25 to 5 μg.mL-1 (comparable to Daktarin®, 2.5 μg.mL-1). Significant differences in AF for adhesive formulations evaluated as a function of immersion time in water were observed (p <0.001), with an upward trend between 1 h and 3 h, followed by reduction or stabilization up to 6 h. The incorporation of NM and polymeric microparticles did not affect the FA of the experimental adhesive and AEG20 showed the best results, with high initial values, and holding them for 6 h. All adhesive formulations showed low or no toxicity (LC50 349.53 to 931.00 μg.mL-1). The proposed denture adhesive formulation was proven compatible with the incorporation of polymeric microparticles containing NM. / A associação de próteses bem adaptadas com a presença tópica de antifúngicos é adequada abordagem terapêutica para estomatite protética (EP). Foi desenvolvido e avaliado um adesivo para prótese removível contendo nitrato de miconazol (NM) incorporado a micropartículas poliméricas muco-adesivas e/ou pH dependentes visando aumento de biodisponibilidade. Inicialmente, foram desenvolvidas micropartículas contendo 10% e 20% de NM, por spray-drying, utilizando os polímeros Gantrez MS-955 (G10, G20), Eudragit L-100 (E10, E20) ou ambos (EG10, EG20). Foi validado um método analítico por cromatografia líquida de alta eficiência (CLAE) para se quantificar NM das micropartículas. Estas foram caracterizadas por microscopia eletrônica de varredura (MEV), difração de raios x, espectrometria de infravermelho por transformada em Fourier (FTIR), calorimetria exploratória diferencial (CED), estudos de liberação in-vitro (porcentagem de dissolução/tempo e perfis de liberação) e atividade antifúngica. A seguir foram desenvolvidas formulações de um adesivo experimental para prótese (ACT) acrescido de 10% em peso das micropartículas (AE1, AG1, AEG1, AE2, AG2, AEG2) ou 2% do fármaco puro (AMN). Para todos adesivos determinou-se: concentração inibitória mínima (CMI) em Candida albicans (microdiluição em caldo e diluição em ágar); força adesiva (FA, entre superfícies acrílicas após 0,5, 1, 3, ou 6 h de imersão em água); e toxicidade em Artemia salina (24 h e 48 h), calculando-se concentração letal 50 (CL50). O método de CLAE apresentou-se específico, linear (r = 0,9992), preciso, exato e robusto na faixa de 5 a 90 μg.mL-1, com tempos de corrida e de retenção de 10,0 e 5,58 minutos, respectivamente. Todas as micropartículas mostraram aceitáveis valores de rendimento (37,22% – 55,36%) e de encapsulação (superiores a 89%). As micropartículas E10 e E20 apresentaram forma esférica e superfície lisa, enquanto EG20 possuíam a mesma forma, porém superfície rugosa. As micropartículas G10, G20 e EG20 apresentaram morfologia deprimida e crateras. Os diâmetros das micropartículas variaram entre 1,9 a 4,3 μm. Nenhuma ligação química foi observada entre o NM e os polímeros, através dos espectros de FTIR. A microencapsulação contribuiu para amorfizar o fármaco, segundo as análises térmicas e difração de raios X, reduzindo seu tempo de liberação. Ajustaram-se ao modelo cinético de liberação biexponencial o NM e as micropartículas G10, G20 e EG20, e ao modelo monoexponencial, as micropartículas E10, E20 e EG10. As micropartículas apresentaram eficiência antifúngica similar ao fármaco puro. Extratos dos adesivos contendo micropartículas e a formulação AMN apresentaram CMI entre 1,25 a 5 μg.mL-1 (comparável a Daktarin®, 2,5 μg.mL-1). Foram verificadas diferenças significativas na FA para as formulações de adesivos avaliadas em função do tempo de imersão na água (p<0,001), com tendência de aumento entre 1 h e 3 h, seguido de decréscimo ou estabilização até 6 h. A incorporação do NM e de micropartículas poliméricas não prejudicou a FA do adesivo experimental e AEG20 exibiu os melhores resultados, apresentando elevados valores iniciais, e mantendo-os por 6 h. Todas as formulações de adesivos apresentaram baixa ou nenhuma toxicidade (CL50 de 349,53 a 931,00 μg.mL-1). A formulação de adesivo para prótese removível proposta foi compatível com a incorporação de micropartículas poliméricas contendo NM.

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.

No description available.

Plusieurs souches cliniques de Candida albicans résistantes aux médicaments antifongiques azolés surexpriment des gènes encodant des effecteurs de la résistance appartenant à deux classes fonctionnelles : i) des transporteurs expulsant les azoles, CDR1, CDR2 et MDR1 et ii) la cible des azoles 14-lanostérol déméthylase encodée par ERG11. La surexpression de ces gènes est due à la sélection de mutations activatrices dans des facteurs de transcription à doigts de zinc de la famille zinc cluster (Zn2Cys6) qui contrôlent leur expression : Tac1p (Transcriptional activator of CDR genes 1) contrôlant l’expression de CDR1 et CDR2, Mrr1p (Multidrug resistance regulator 1), régulant celle de MDR1 et Upc2p (Uptake control 2), contrôlant celle d’ERG11. Un autre effecteur de la résistance clinique aux azoles est PDR16, encodant une transférase de phospholipides, dont la surexpression accompagne souvent celle de CDR1 et CDR2, suggérant que les trois gènes appartiennent au même régulon, potentiellement celui de Tac1p. De plus, la régulation transcriptionnelle du gène MDR1 ne dépend pas seulement de Mrr1p, mais aussi du facteur de transcription de la famille basic-leucine zipper Cap1p (Candida activator protein 1), un régulateur majeur de la réponse au stress oxydatif chez C. albicans qui, lorsque muté, induit une surexpression constitutive de MDR1 conférant la résistance aux azoles. Ces observations suggèrent qu’un réseau de régulation transcriptionnelle complexe contrôle le processus de résistance aux antifongiques azolés chez C. albicans. L’objectif de mon projet au doctorat était d’identifier les cibles transcriptionnelles directes des facteurs de transcription Tac1p, Upc2p et Cap1p, en me servant d’approches génétiques et de génomique fonctionnelle, afin de i) caractériser leur réseau transcriptionnel et les modules transcriptionnels qui sont sous leur contrôle direct, et ii) d’inférer leurs fonctions biologiques et ainsi mieux comprendre leur rôle dans la résistance aux azoles. Dans un premier volet, j’ai démontré, par des expériences de génétique, que Tac1p contrôle non seulement la surexpression de CDR1 et CDR2 mais aussi celle de PDR16. Mes résultats ont identifié une nouvelle mutation activatrice de Tac1p (N972D) et ont révélé la participation d’un autre régulateur dans le contrôle transcriptionnel de CDR1 et PDR16 dont l’identité est encore inconnue. Une combinaison d’expériences de transcriptomique et d’immunoprécipitation de la chromatine couplée à l’hybridation sur des biopuces à ADN (ChIP-chip) m’a permis d’identifier plusieurs gènes dont l’expression est contrôlée in vivo et directement par Tac1p (PDR16, CDR1, CDR2, ERG2, autres), Upc2p (ERG11, ERG2, MDR1, CDR1, autres) et Cap1p (MDR1, GCY1, GLR1, autres). Ces expériences ont révélé qu’Upc2p ne contrôle pas seulement l’expression d’ERG11, mais aussi celle de MDR1 et CDR1. Plusieurs nouvelles propriétés fonctionnelles de ces régulateurs ont été caractérisées, notamment la liaison in vivo de Tac1p aux promoteurs de ses cibles de façon constitutive et indépendamment de son état d’activation, et la liaison de Cap1p non seulement à la région du promoteur de ses cibles, mais aussi celle couvrant le cadre de lecture ouvert et le terminateur transcriptionnel putatif, suggérant une interaction physique avec la machinerie de la transcription. La caractérisation du réseau transcriptionnel a révélé une interaction fonctionnnelle entre ces différents facteurs, notamment Cap1p et Mrr1p, et a permis d’inférer des fonctions biologiques potentielles pour Tac1p (trafic et la mobilisation des lipides, réponse au stress oxydatif et osmotique) et confirmer ou proposer d’autres fonctions pour Upc2p (métabolisme des stérols) et Cap1p (réponse au stress oxydatif, métabolisme des sources d’azote, transport des phospholipides). Mes études suggèrent que la résistance aux antifongiques azolés chez C. albicans est intimement liée au métabolisme des lipides membranaires et à la réponse au stress oxydatif. / Many azole resistant Candida albicans clinical isolates overexpress genes encoding azole resistance effectors that belong to two functional categories: i) CDR1, CDR2 and MDR1, encoding azole-efflux transporters and ii) ERG11, encoding the target of azoles 14-lanosterol demethylase. The constitutive overexpression of these genes is due to activating mutations in transcription factors of the zinc cluster family (Zn2Cys6) which control their expression. Tac1p (Transcriptional activator of CDR genes 1), controlling the expression of CDR1 and CDR2, Mrr1p (Multidrug resistance regulator 1), regulating MDR1 expression and Upc2p (Uptake control 2), controlling the expression of ERG11. Another determinant of clinical azole resistance is PDR16, encoding a phospholipid transferase, whose overexpression often accompanies that of CDR1 and CDR2 in clinical isolates, suggesting that the three genes belong to the same regulon, potentially that of Tac1p. Further, MDR1 expression is not only regulated by Mrr1p, but also by the basic-leucine zipper transcription factor Cap1p (Candida activator protein 1), which controls the oxidative stress response in C. albicans and whose mutation confers azole resistance via MDR1 overexpression. These observations suggest that a complex transcriptional regulatory network controls azole resistance in C. albicans. My Ph.D. studies are aimed at identifying the direct transcriptional targets of Tac1p, Upc2p and Cap1p using genetics and functional genomics approches in order to i) characterize their regulatory network and the transcriptional modules under their direct control and ii) infer their biological functions and better understand their roles in azole resistance. In the first part of my studies, I showed that Tac1p does not only control the expression of CDR1 and CDR2, but also that of PDR16. My results also identified a new activating mutation in Tac1p (N972D) and revealed that the expression of CDR1 and PDR16 is under the control of another yet unknown regulator. The combination of transcriptomics and genome-wide location (ChIP-chip) approaches allowed me to identify the in vivo direct targets of Tac1p (PDR16, CDR1, CDR2, ERG2, others), Upc2p (ERG11, ERG2, MDR1, CDR1, others) and Cap1p (MDR1, GCY1, GLR1, others). These results also revealed that Upc2p does not only control the expression of ERG11 but also that of MDR1 and CDR1. Many new functional features of these transcription factors were found, including the constitutive binding of Tac1p to its targets under both activating and non-activating conditions, and the binding of Cap1p which extends beyond the promoter region of its target genes, to cover the open reading frame and the putative transcription termination regions, suggesting a physical interaction with the transcriptional machinery. The characterization of the transcriptional regulatory network revealed a functional interaction between these factors, notably between Cap1p and Mrr1p, and inferred potential biological functions for Tac1p (lipid mobilization and traffic, response to oxidative and osmotic stress) and confirmed or suggested other functions for Upc2p (sterol metabolism) and Cap1p (oxidative stress response, regulation of nitrogen utilization and phospholipids transport). Taken together, my results suggest that azole resistance in C. albicans is tightly linked to membrane lipid metabolism and oxidative stress response.

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.

Made available in DSpace on 2014-12-17T14:16:35Z (GMT). No. of bitstreams: 1 WalicyranisonPS_DISSERT.pdf: 2780035 bytes, checksum: 20a70f53e533bfb0816b6d9b02b8af32 (MD5) Previous issue date: 2013-06-11 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Candida albicans is a diploid yeast that in some circumstances may cause oral or oropharyngeal infections. The investigation of natural products is mandatory for the discovery of new targets for antifungal drugs development. This study aimed to determine the genotypes of 48 clinical isolates of C. albicans obtained from the oral cavity of kidney transplant patients from two distinct geographic regions of Brazil. In addition, we investigated three virulence factors in vitro: phospholipase activity, morphogenesis and the ability to evade from polymorphonuclear neutrophils. The expression of these virulence factors in vitro was also investigated in the presence of the crude extract of Eugenia uniflora. The genotype A was the most prevalent (30 isolates; 62.5%), followed by genotype C (15 isolates; 31.5%) and genotype B (3 isolates; 6.25%). When microsatellite technique with primer M13 was applied, 80% of the isolates from the South were placed within the same cluster. All Genotype C strains were grouped together within two different clusters. Genotype C was considered more resistant to PMNs attack than genotypes A and B. Strains isolated from the South of Brazil showed higher ability to combat PMNs phagocytosis. We found a high rate of genotype C strains isolated from the oral cavity of this group of patients. The crude extract of E. uniflora inhibited proper hypha formation and phagocytosis by PMNs, but had no significant effect on phospholipase activity. This study characterized oral C. albicans strains isolated from kidney transplant recipients and will contribute for the better understanding of the pathogenesis and alternative therapeutics for oral candidiasis / Candida albicans ? uma levedura dipl?ide que em certas circunst?ncias pode causar infec??es da cavidade oral e da orofaringe. A investiga??o de produtos naturais ? fundamental para a descoberta de novos alvos para o desenvolvimento de drogas antif?ngicas. Este estudo objetivou determinar os gen?tipos de 48 isolados cl?nicos de C. albicans obtidos da cavidade oral de pacientes transplantados renais de duas distintas regi?es geogr?ficas do Brasil. Al?m disso, foram investigados tr?s fatores de virul?ncia in vitro: atividade de fosfolipase, morfog?nese e a capacidade de escapar do ataque de neutr?filos polimorfonucleares. A express?o destes fatores de virul?ncia tamb?m foi investigada na presen?a do extrato bruto de Eugenia uniflora. O gen?tipo A foi o mais prevalente (30 isolados; 62,5%), seguido do gen?tipo C (15 isolados; 31,5%) e do gen?tipo B (3 isolados; 6,25%). Quando a t?cnica do microssat?lite com o primer M13 foi empregada, 80% dos isolados da regi?o Sul foram agrupados no mesmo cluster. Todos os isolados do gen?tipo C foram agrupados juntos em dois diferentes clusters bem definidos. Isolados do gen?tipo C foram considerados mais resistentes ? a??o de PMNs do que os dos gen?tipos A e B. As cepas isoladas do Sul do Brasil demonstraram maior habilidade em combater a fagocitose por PMNs. Encontrou-se uma alta taxa de isolados do gen?tipo C da cavidade oral deste grupo de pacientes. O extrato bruto de E. uniflora inibiu a forma??o de hifa e fagocitose por PMNs, mas n?o apresentou efeito significativo na atividade de fosfolipase. Este estudo caracterizou isolados cl?nicos de C. albicans da cavidade oral de pacientes transplantados renais, contribuindo para um melhor entendimento da patog?nese e terap?utica alternativa para a candid?ase oral

Uma infecção pulpar pode resultar na colonização microbiana de todo sistema de canais radiculares incluindo os túbulos dentinários. Estes microorganismos e seus produtos tóxicos são responsáveis pelo desenvolvimento e persistência da periodontite apical de origem endodôntica. O presente estudo objetivou avaliar a viabilidade de E. faecalis e C. albicans em túbulos dentinários após a aplicação de hidróxido de cálcio, clorexidina gel 2%, hidróxido de cálcio associado à clorexidina gel 2% e soro fisiológico, através da análise por cultura microbiológica e microscopia de fluorescência. Para tanto 120 raízes de dentes humanos foram padronizadas e autoclavadas, sendo posteriormente divididas em 2 grupos (n= 60) para contaminação com E. faecalis e C. albicans por 21 dias. Em seguida, foram divididas em 8 grupos (n= 15) para aplicação das substâncias antimicrobianas nos canais radiculares e posterior incubação em estufa por 14 dias. Amostras da dentina radicular na extensão de 0 - 100 µm e de 100 - 200 µm foram coletadas e submetidas à cultura microbiológica através do plaqueamento em meios de cultura. Após 48 horas de incubação promoveu-se a avaliação das UFC. Paralelamente, as amostras foram processadas para análise em microscopia de fluorescência com auxílio de marcadores fluorescentes específicos, a fim de se determinar a proporção de microorganismos viáveis e não viáveis. Outros 6 espécimes foram preparados para análise em MEV. Os resultados mostraram uma maior capacidade de penetração intratubular para E. faecalis quando comparado a C. albicans. A aplicação de medicação intracanal resultou em significativa redução da viabilidade dos microorganismos quando comparado ao grupo controle independente da medicação aplicada e em ambas as porções da dentina radicular avaliadas. Entretanto, ao compararmos individualmente as medicações, observamos o melhor desempenho da clorexidina gel 2 % e da associação de hidróxido de cálcio e clorexidina gel 2% sem diferença significante entre elas. O hidróxido de cálcio apresentou os piores resultados para desinfecção dos canais radiculares contaminados com E. faecalis e C. albicans. Estes achados foram confirmados tanto pela cultura microbiológica quanto pela microscopia de fluorescência. A cultura microbiológica e a microscopia de fluorescência são métodos adequados e complementares para avaliação da viabilidade de E. faecalis e C. albicans e a eficácia da clorexidina gel 2% e da associação hidróxido de cálcio e clorexidina gel 2% justificam seu uso em endodontia como medicação intracanal. / A pulp infection can result in a microbial colonization of the entire root canals system, including dentinal tubules. These microorganisms and their toxic product are responsible for the development and persistence of apical periondontitis from endodontic source. The present study aimed to evaluate E. faecalis and C. albicans viability in dentinal tubules after the application of calcium hydroxide, chlorhexidine gel 2%, calcium hydroxide associated to chlorhexidine gel 2% and physiological solution, through the analysis by microbiological culture and fluorescence microscopy. For that, 120 human teeth root were standardized and submitted to autoclave, and after ere divided into 2 groups (n=60) for E. faecalis and C. albicans contamination for 21 days. Following this, they were divided into 8 groups (n=15) for application of antimicrobial substances in the root canals and subsequent incubation for 14 days. Samples from root dentin with 0 - 100 µm and 100 - 200 µm of extension were collected and submitted to microbiological culture.After 48 hours of incubation, it was performed the evaluation of colony-forming units (CFU). At the same time, the samples were processed for fluorescence microscopic analysis with the assistance of specific fluorescent markers, in order to determine the proportion of viable and non-viable microorganisms. Other 6 specimens were prepared for the analysis of scanning electron microscopy. Results demonstrated a higher capacity of penetration in the tubules for E. faecalis than for C. albicans. The application of intracanal medication resulted in significant reduction of microorganisms viability when compared to the control groups independently of the medication used and in both evaluated portions of root dentin. However, when one compares individually the medications, it was observed a better performance of chlorhexidine gel 2% and the association of calcium hydroxide with chlorhexidine gel 2% without a significant difference between them. Calcium hydroxide had the worst results for disinfection of root canal contaminated with E. faecalis and C. albicans. These findings were confirmed for the microbiological culture as well as for the fluorescence microscopy. The microbiological culture and the fluorescence microscopy are adequate methods and complementary for the evaluation of E. faecalis and C. albicans viability and the efficacy of chlorhexidine gel 2% and the association of calcium hydroxide with chlorhexidine gel 2% warrant their use in Endodontics as an intracanal medication.

O antisséptico cloreto de cetilpiridíneo (CCP) demonstrou ser eficaz como alternativa terapêutica aos antifúngicos em candidose oral, porém, apresenta baixa substantividade. Para aumentar sua biodisponibilidade surge a possibilidade de incorporá-lo num sistema de liberação lenta composto por filmes poliméricos nanofibrílicos. O objetivo deste estudo foi preparar filmes nanofibrílicos através de eletrofiação e incorporar CCP, fazer sua caracterização térmica e morfológica, além de análise microbiológica dos filmes contendo CCP frente a cepas de Candida albicans. O fármaco CCP foi adicionado a três soluções poliméricas, de álcool polivinílico (PVA), polivinilpirrolidona (PVP) e a de PVP/PM [Poli (metacrilato de metila-co-acrilato de etila-co-metacrilato de amônio)] em 5% de concentração. As soluções foram avaliadas quanto à condutividade, viscosidade e tensão superficial e, posteriormente, passaram pelo processo de eletrofiação para obtenção de filmes nanofibrílicos. Os filmes nanofibrílicos foram caracterizados através de análise morfológica (microscopia eletrônica de varredura - MEV), análise térmica (análise termogravimétrica - TGA e calorimetria exploratória diferencial - DSC) e análise química (espectro de infravermelho da transformada de Fourier - FTIR). A eficiência de encapsulação de CCP nas nanofibras, o perfil cinético de liberação das nanofibras e a permeação em mucosa suína (células de Franz) foram avaliados por espectrofotometria. Foi ainda avaliada a concentração de CCP a ser incorporada (de 0,05 a 5%) em nanofibra para que a fração liberada seja a mínima fungicida frente às cepas de C. albicans susceptíveis, pela técnica de disco-difusão, comparada ao miconazol 5% (MCZ). Nestas condições, a solução de PVA apresentou a maior condutividade, viscosidade e tensão superficial. CCP aumentou a condutividade. Ambos, CCP e PM não alteram a tensão superficial do PVP. PM reduziu a viscosidade. A solução de PVP/PM formou o filme mais uniforme e com menos beads. Os termogramas (TGA/DSC) sugerem que o processo de eletrofiação diminiu a cristalinidade de CCP. A eficiência de encapsulação de CCP alcança 99%. A taxa de liberação de CCP encapsulado foi lenta durante o período experimental de 24h. Quando em filme nanofibrílicos, a atividade fungicida de CCP 2,5% e de MCZ 5% foi similar, mas somente MCZ foi fungistático. O filme nanofibrílico contrai em contato com o meio de cultura. Pode-se concluir que filmes nanofibrílicos de PVP/PM com CCP tem potencial para uso como alternativa para tratamento de infecções orais causadas por Candida. / The aim of this study was to prepare electrospun nanofiber films cetylpyridinium chloride (CPC) incorporated test their potential use as antifungal therapy against Candida albicans. CPC was incorporated to three different polymeric solutions, one containing polyvinyl alcohol (PVA), the second containing polyvinylpyrrolidone (PVP) and the third prepared with PVP and poly (methyl methacrylate-co-ethyl acrylate-co-methacrylate ammonium) (PMMA). These polymeric solutions were evaluated as to conductivity, viscosity and surface tension, then subjected to a electrospinning process to obtain nanofiber films. The morphology and structure of nanofiber films were analyzed using scanning electron microscopy (SEM). The characterization of nanofiber films was performed by thermogravimetric analysis (TG) and differential scanning calorimetry (DSC). The encapsulation efficiency of the CPC nanofibers and the release kinetic profile of CPC and porcine mucosa permeation (Franz cells) were assessed by spectrophotometry. It was also assessed the concentration of CPC to be incorporated (from 0.05 to 5%) in nanofiber to be minimal fungicidal fraction released against strains of C. albicans by disk diffusion tests, compared to 5% miconazole (MCZ). Under these conditions, the PVA solution showed the highest conductivity, viscosity and surface tension. CPC increased conductivity. Both CPC and PMMA did not alter the surface tension of PVP. PMMA reduced viscosity. The solution of PVP/PMMA formed a more uniform film with less beads. Thermograms (TGA / DSC) suggested that the electrospinning process changes the crystallinity of CPC. The encapsulation efficiency reaches 99% of CPC. The release rate of encapsulated CPC was slow during the experimental period of 24 hours. In a nanofibrilic film, the fungicidal activity of CPC 2.5% and 5% MCZ was similar, but only MCZ proved fungistatic action. The nanofibrilic film shows contraction in contact with the culture medium. It can be concluded that nanofibrilic films PVP/PMMA with CPC has potential for use as an alternative for the treatment of oral infections caused by Candida.