published_or_final_version / Dentistry / Doctoral / Doctor of Philosophy

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Microscopical examination of <I>Candida albicans</I> grown on contoured artificial surfaces provided evidence that hyphae responded thigmotropically to features of the growth substrate. Hyphae of <I>C. albicans</I> followed grooves and ridges on various artificial membranes and penetrated pores of Nucleopore filters. The thigmotropic response in <I>C. albicans</I> was attenuated by gadolinium ions and by verapamil suggesting that calcium uptake may be involved in thigmotropic regulation. Thigmotropism was also observed for the first time in three genera of dermatophytic fungi <I>(Epidermophyton, Trichophyton </I>and <I>Microsporum)</I> and two saprophytic fungi <I>(Mucor mucedo </I>and <I>Neurospora crassa</I>). Therefore thigmotropism may be a general feature of fungal hyphae that must forage for nutrients on surfaces and within solid materials. Since Ca²⁺ appears to be involved in the regulation of thigmotropism attempts were made to construct strains expressing the Ca²⁺sensitive photoprotein aequorin. The apoaequorin d gene was cloned in to <I>C. albicans</I> and <I>S. cerevisiae</I> using the YPB-ADHpt expression vector. Southern analysis indicated low copy number of the plasmid in <I>C. albicans</I> as compared with <I>S. cerevisiae. </I>Aequorin was reconstituted in protein extracts of <I>C. albicans</I> and <I>S. cerevisiae</I> by supplementing them with coelenterazine. The level for <I>C. albicans </I>was ten times higher than for <I>Neurospora crassa</I>, the only filamentous fungus to be transformed with this gene so far. Aequorin was successfully reconstituted in transformed living cells, and the luminescence levels were sufficiently high to be detected when external Ca²⁺ was added to the growth medium. Transformed <I>C. albicans</I> cells undergoing the dimorphic transition from yeast-to-hyphal form exhibited higher resting levels of luminescence indicating that cells induced to form hyphae have higher [Ca²⁺] than yeast cells. The work presented in this thesis presents first evidence of construction of strains expressing the luminescence photoprotein aequorin in a pathogenic fungus. This method provides a non-toxic, non-invasive method for monitoring [Ca²⁺] in <I>C. albicans.</I>

Trabajo de Investigación Requisito para optar al Título de Cirujano Dentista / Autor no autoriza el acceso a texto completo de su documento / INTRODUCCIÓN: La diabetes mellitus tipo 2 (DM2) es un trastorno metabólico debido a una deficiencia progresiva en la secreción de insulina. El control glicémico en pacientes con DM2 se debe mantener en niveles adecuados; si estos no se mantienen, pueden presentarse consecuencias negativas locales y sistémicas importantes. El examen de Hemoglobina Glicosilada (HbA1C) se utiliza para definir el verdadero control glicémico de la enfermedad, por lo que debe mantenerse en niveles adecuados. Las levaduras del género Candida son microorganismos comensales, que cambian a una forma patógena al existir alteración en los mecanismos de defensa del hospedero. La DM2, especialmente en estado descompensado, se ha asociado al desarrollo de patologías provocadas por infección con microorganismos oportunistas, en especial por levaduras del género Candida. MATERIAL Y MÉTODOS: Se obtuvieron muestras de saliva de 52 sujetos voluntarios mayores de 30 años con diagnóstico de DM2, previo consentimiento informado firmado. El grupo se dividió según su condición metabólica en diabéticos compensados (HbA1C < 7%) y no compensados (HbA1C > 7%). Un volumen de 100 μl de saliva se sembró en placas de Agar Sabouraud Tetraciclina y CHROMagar Candida ®, las que se incubaron durante 48 horas a 30 ºC, hasta el desarrollo de colonias. En estas placas se estimó el número de células viables y se identificaron presuntivamente las especies de Candida presente según su coloración. La identidad de las colonias observadas se confirmó molecularmente mediante Reacción en Cadena de la Polimerasa (PCR) y bioquímicamente mediante el Sistema de Identificación API ID32C ®. RESULTADOS: El promedio de HbA1C de la muestra total correspondió a 8,16 %, donde 40 correspondieron a mujeres y 12 a hombres, entre 33 y 82 años de edad. La portación de levaduras no tuvo diferencias significativas en relación al género y al estado metabólico (p > 0,05) pero fue mayor al aumentar la edad (p = 0,024). La cantidad de UFC, medida en rangos mayor o menor a 400 UFC/ml fue mayor en el 6 grupo descompensado (p = 0,025) y al aumentar la edad (p = 0,017). Se identificaron 6 especies, donde la más aislada en la muestra fue C. albicans con un 66,1 % y en 38 (97,4 %) del total de sujetos portadores, seguido por C. glabrata. La diversidad de especies no se relacionó a género ni al estado metabólico (p > 0,05) pero sí a la edad (p = 0,049). CONCLUSIONES: La descompensación metabólica en pacientes con DM2 se asoció a una mayor cantidad de UFC/ml no así con una mayor diversidad de especies.

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Cao Boyang. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references. / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web.

Orientador: Juliana Campos Junqueira / Banca: Célia Regina Gonçalves e Silva / Banca: Antonio Olavo Cardoso Jorge / Resumo: Leveduras do gênero Candida são patógenos oportunistas que podem causar infecções superficiais ou sistêmicas. A investigação de plantas medicinais contra Candida spp. tem aumentado significativamente nos últimos anos. O presente estudo teve como objetivos avaliar a ação antimicrobiana do extrato hidroalcoólico e do óleo essencial de Mentha piperita L. sobre isolados clínicos de Candida spp. e observar o efeito da infusão de Mentha piperita L. sobre a aderência de leveduras do gênero Candida à resina acrílica. O extrato hidroalcoólico e o óleo essencial de Mentha piperita L. foram avaliados quanto à atividade antimicrobiana sobre diferentes espécies do gênero Candida, sendo cinqüenta amostras de C. albicans, dez C. glabrata, dez C. tropicalis, oito C. parapsilosis e duas C. krusei. A concentração inibitória mínima (CIM) e concentração fungicida mínima (CFM) do extrato hidroalcoólico (500 g de planta fresca/1 L de etanol) foram determinadas pelo método da diluição em caldo. O grupo controle constituiu-se de etanol na mesma concentração do extrato (79°GL). A atividade antimicrobiana do óleo essencial foi realizada por meio do teste de difusão em ágar. Foi observado o efeito da infusão de Mentha piperita L. sobre a aderência de cepas padrão de C. albicans, C. glabrata, C. tropicalis e C. krusei à resina acrílica. Os resultados demonstraram que o extrato hidroalcoólico de Mentha piperita L. apresentou atividade fungistática para algumas cepas de C. albicans, C. tropicalis e C. glabrata e atividade fungicida sobre cepas de todas as espécies de Candida estudadas em relação ao grupo controle. O óleo essencial mostrou maior atividade inibitória para cepas de C. albicans, seguida por C. tropicalis e C. parapsilosis. A infusão de Mentha piperita L. não foi capaz... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Fungi belonging to Candida species are opportunistic pathogens that can cause local and systemic infections. The investigation of natural products against Candida spp. increased significantly in the last few years. In this present study, the ethanolic extract and essential oil from Mentha piperita L. were evaluated for antimicrobial activity against Candida spp., being fifty strains from C. albicans, ten from C. glabrata, ten from C. tropicalis, eight from C. parapsilosis and two C. krusei. The minimal inhibition concentration (MIC) and minimal fungicidal concentration (MFC) of the ethanolic extract (500 g of fresh plant / 1 L of ethanol) were determined by the broth dilution method. The control group was constituted by ethanol in the same proportion of the extract (79ºGL). The antimicrobial activity of the essential oil was performed by the agar diffusion method. Besides, it was observed the effect of Mentha piperita L. infusion on the adherence of standard strains of C. albicans, C. glabrata, C. tropicalis and C. krusei to the acrylic resin. The results showed that ethanolic extract of Mentha piperita L. presented fungistatic activity to some strains of C. albicans, C. glabrata and C. tropicalis, and also fungicidal activity on strains of all Candida species in comparison with the control group. The essential oil of Mentha piperita L.was more active to strains of C. albicans followed by C. tropicalis and C. parapsilosis. The infusion of Mentha piperita L. was not effective to inhibit the Candida adherence to the acrylic resin. It was concluded that Mentha piperita L. was more active to strains of C. albicans and C. tropicalis, but was not effective to have any effect on the adherence of these microorganisms to the acrylic resin. / Mestre

Der Hefepilz Candida albicans gehört zu den fakultativ pathogenen Infektionserregern und ist Teil der natürlichen Mikroflora der Schleimhäute des Verdauungs- und Urogenitaltraktes der meisten gesunden Menschen. Ist das Gleichgewicht der Flora gestört, kann es zu oberflächlichen Mykosen kommen, wie z.B. der oropharyngealen Candidiasis (Mundsoor), die in der Regel durch die Gabe eines Antimykotikums in wenigen Tagen zu behandeln sind. In seltenen Fällen kann es auch zu schwerwiegenden Infektionsverläufen bis hin zu lebensbedrohlichen systemischen Mykosen kommen. Hauptsächlich immunsupprimierte Patienten, wie z.B. AIDS-Patienten oder Personen, die kürzlich einer Organ- oder Knochenmarkstransplantation unterzogen wurden, leiden häufig an oberflächlichen C. albicans-Infektionen. Insbesondere bei wiederkehrenden Infektionen ist der Pilz in der Lage, gegen das häufig verabreichte Medikament Fluconazol eine Resistenz zu entwickeln. Ein wichtiger Mechanismus dieser Resistenzentwicklung ist die Überexpression von Effluxpumpen, die das Medikament aus der Zelle heraustransportieren. Zwei Arten von Effluxpumpen, die eine Rolle in der Resistenzentwicklung in C. albicans spielen, konnten bisher identifiziert werden, die ABC (ATP binding cassette)-Transporter Cdr1 und Cdr2 sowie der MFS (major facilitator superfamily)-Transporter Mdr1. Der Zinc-Cluster Transkriptionsfaktor Mrr1 spielt eine wichtige Rolle in der Regulation der MDR1-E¬ffluxpumpe. Er kontrolliert die MDR1-Expression in Anwesenheit induzierender Substanzen und sogenannte "gain-of-function" Mutationen in MRR1 konnten als die Ursache der konstitutiven MDR1-Hochregulierung und der "Multidrug-Resistance" in C. albicans identifiziert werden. In dieser Arbeit konnte ein Ortholog zu MRR1 aus C. albicans in Candida dubliniensis, einer zu C. albicans nahe verwandten Hefe, identifiziert werden. Es wurde gezeigt, dass in den untersuchten klinischen und in vitro generierten Fluconazol-resistenten C. dubliniensis-Stämmen ebenfalls gain-of-funcion Mutationen in MRR1 die MDR1-Überexpression und eine Resistenz bewirken. Die Ergebnisse demonstrieren, dass der Transkriptionsfaktor Mrr1 eine wichtige Rolle in der Entwicklung der Resistenz in diesen humanpathogenen Pilzen spielt. Bisher ist nicht bekannt, wie der Zinc-Cluster Transkriptionsfaktor MRR1 durch induzierende Substanzen oder gain-of-function Mutationen aktiviert wird. Um zu verstehen, wie die Mrr1- Aktivität reguliert wird, wurden in dieser Arbeit durch Deletionsstudien funktionelle Domänen des Transkriptionsfaktors identifiziert. Um einen besseren Einblick in die Regulation der MDR1-vermittelten Resistenz in C. albicans zu bekommen, wurde in dieser Arbeit die gegenseitige Abhängigkeit von Mrr1 und Cap1 bzw. Upc2 in Bezug auf die MDR1-Expression untersucht. Es wurden ChIP-on-chip Analysen und Transkriptionsprofile mit aktiviertem Mrr1 durchgeführt, um direkte Targets von Mrr1 zu identifizieren. Mit der vorliegenden Arbeit wurde ein wichtiger Beitrag zum Verständnis der Entwicklung der Multidrug-Resistenz in C. albicans geleistet. E¬ffluxpumpen und deren Regulatoren stellen in der Bekämpfung von C. albicans-Infektionen ein interessantes Angriffsziel für die Entwicklung neuer Medikamente und die Weiterentwicklung bereits vorhandender Antimykotika dar. / The yeast Candida albicans is a human fungal pathogen and is part of the microflora of mucosal surfaces of the gastrointestinal and urogenital tract in most healthy people. If the balance of the flora is disturbed C. albicans can cause super cial mycoses, e.g. oropharyngeal Candidiasis, also known as "thrush", which are usually easy to cure within a few days by treatment with antimycotic drugs. Infections with the yeast can also result in serious as well as life-threatening systemic mycoses. However, immunocompromised patients, e.g. AIDS patients, often suffer from super cial C. albicans infections and especially in recurrent infections the yeast can develop resistance to the commonly used antifungal drug fluconazole. An important mechanism of drug resistance is the overexpression of e¬ux pumps, which mediate the transport of toxic compounds out of the cell. Two types of e¬fflux pumps, which play a role in die development of resistance in C. albicans, have been described so far, the ABC (ATP binding cassette) transporters Cdr1 and Cdr2, and the MFS (major facilitator superfamily) transporter Mdr1. The zinc cluster transcription factor Mrr1 plays an important role in the regulation of the MDR1 gene. It controls the MDR1 expression in response to inducing chemicals and gain-of function mutations in MRR1 are responsible for the constitutive upregulation of MDR1 and fluconazole resistance. In this work a CaMRR1 ortholog was found in Candida dubliniesis, a yeast closely related to C. albicans. It could be shown that gain-of-function mutations in CdMRR1 were the cause of MDR1 overexpression and drug resistance in all investigated clinical and in vitro generated strains. The results showed that Mrr1 plays an important role in the development of drug resistence in these human fungal pathogens. Currently it is not understood how these zinc cluster transcription factors are activated under inducing conditions or by gain-of-function mutations. To better understand the regulation of Mrr1 activation, in this work deletion studies were performed to identify functional domains of the transcription factor. To gain better insight into the regulation of MDR1-mediated drug resistance in C. albicans, the interdependence of Mrr1 and two other MDR1 regulators, Cap1 and Upc2, was studied in this work. ChIP-on-chip analyses and transcriptional profiles with acitvated Mrr1 were performed to identify direct targets of Mrr1. This thesis contributes to the understanding of the development of multidrug resistance in C. albicans. Efflux pumps and their transcriptional regulators provide an interesting target for the development of new antifungal drugs or the further development of available drugs against C. albicans infections.

Dissertation--Biologie--Berlin--Technische Universität, 2003. / Bibliogr. p. 101-116.