Düsseldorf, Universiẗat, Diss., 2004. / Erscheinungsjahr an der Haupttitelstelle: 2003.

Düsseldorf, Universiẗat, Diss., 2001.

Düsseldorf, Universiẗat, Diss., 2001.

Thesis (Ph.D.)--University of Nebraska-Lincoln, 2007. / Title from title screen (site viewed July 9, 2007). PDF text: 87 p. : ill. UMI publication number: AAT 3252833. Includes bibliographical references. Also available in microfilm and microfiche formats.

Trabajo de Investigación Requisito para optar al Título de Cirujano Dentista / Autor no autoriza el acceso a texto completo de su documento / Existe controversia en los resultados obtenidos por investigaciones que valoran la diversidad genética de levaduras de sitios subgingivales sanos y enfermos. Recientemente, un estudio concluyó que las levaduras orales aisladas de sujetos enfermos son genéticamente más diversas que las de grupos con salud oral. El propósito de este trabajo fue medir la variabilidad genética de 103 aislados de levaduras de sujetos periodontalmente sanos. Para ello, se les extrajo ADN genómico, el cual fue analizado por la técnica de amplificación al azar de ADN polimórfico, mediante reacción en cadena de la polimerasa (RAPD-PCR). Se determinó que los aislados de levaduras de mucosa y subgingiva sanas son genéticamente homogéneos dentro de los individuos, con mantención de cepas y microevolución. Sin embargo, entre grupos de aislados de sujetos diferentes existe heterogeneidad genética. El S AB promedio calculado para mucosa y subgingiva de todos los aislados fue de 0,49. El análisis de todos los aislados permitió determinar 55 genotipos diferentes. Los genotipos más frecuentes fueron el genotipo 6, que lo presentaron 9 de 11 aislados del sujeto C6 y el genotipo 19, que estaba presente en 3 de 4 aislados del sujeto CD. La mayor diversidad genotípica (G) la presentó el sujeto C23 (G=15,2). De esta forma, se ha caracterizado genéticamente un grupo de aislados control, que proviene de mucosa y sitios subgingivales sanos, que en el futuro permitirá realizar el mismo tipo de análisis en levaduras de pacientes con enfermedad periodontal, ya sea gingivitis, periodontitis agresiva o periodontitis crónica.

Orientador: Juliana Campos Junqueira / Banca: Antonio Olavo Cardoso Jorge / Banca: Luis Vitor Silva do Sacramento / Resumo: O objetivo desse estudo foi avaliar a atividade antimicrobiana in vitro e in vivo dos óleos essenciais de Cymbopogon citratus e Melaleuca alternifolia sobre Candida albicans. No estudo in vitro foi determinada a Concentração Inibitória Mínima (CIM) de M. alternifolia e C. citratus em culturas planctônicas de C. albicans de acordo com as normas da NCCLS. Os efeitos dos óleos essenciais também foram testados sobre biofilmes de C. albicans formados no fundo da placa de 96 poços por 48 horas. Para a realização do estudo in vivo, foram utilizados 42 camundongos imunossuprimidos por injeções de prednisolona. A indução de candidose experimental foi realizada por inoculações de C. albicans (108 células/mL) no dorso da língua dos animais e administração de tetraciclina na água de beber. A seguir, os camundongos foram tratados com 3 aplicações do óleo essencial de C. citratus, M. alternifolia ou solução fisiológica (grupo Controle) no dorso da língua. Foram realizadas coletas de amostras bucais, antes e após o tratamento, para recuperação de leveduras da cavidade bucal dos animais e contagem de UFC/mL. Após 5 dias da indução da candidose experimental, os animais foram submetidos à eutanásia para análise histológica e de microscopia eletrônica do dorso da língua. Os resultados foram analisados estatisticamente pelos testes de Tukey e Kruskal-Wallis (p ≤ 0,05). Os resultados obtidos nos testes realizados in vitro demonstraram que a CIM foi de 0,195% para M. alternifolia e menor que 0,01% para C. citratus. A redução microbiana observada nos biofilmes de C. albicans foi de 6,88 e 5,84 log, respectivamente, para M. alternifolia e C. citratus. Em relação à recuperação de C. albicans da cavidade bucal dos animais... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The aim of this study was to evaluate the antimicrobial activity in vitro and in vivo of essential oils of Cymbopogon citratus and Melaleuca alternifolia on Candida albicans. In vitro study was determined the minimum inhibitory concentration (MIC) of M. alternifolia and C. citratus in planktonic cultures of C. albicans according to NCCLS standards. The effects of essential oils were also tested on biofilms of C. albicans formed deep inside the 96-well plate for 48 hours. For the study in vivo, we used 42 mice immunosuppressed by injections of prednisolone. The induction of experimental candidiasis was performed by inoculation of C. albicans (108 cells / mL) on the back of the tongue of animals and administration of tetracycline in drinking water. Next, the mice were treated with three applications of the essential oil of C. citratus, M. alternifolia or saline (control group) on the back of the tongue. Samples were collected with mouth before and after treatment for recovery of yeasts from the oral cavity of animals and number of CFU / mL. 5 days after induction of experimental candidiasis, the animals were euthanized for histological and electron microscopy of the tongue. The results were statistically analyzed by Tukey's test and Kruskal-Wallis test (p ≤ 0.05). The results obtained in in vitro tests showed that the MIC was 0.195% for M. alternifolia and less than 0.01% for C. citratus. The microbial reduction observed in biofilms of C. albicans was 6.88 and 5.84 log, respectively, for M. alternifolia and C. citratus. Regarding the recovery of C. albicans from the oral cavity of animals, there was a reduction of 6.49 log for microbial M. alternifolia and 5.95 for log C. citratus. In the microscopic analysis of the tongue, there was... (Complete abstract click electronic access below) / Mestre

Orientador: Alicia Juliana Kowaltowski / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-08T16:58:37Z (GMT). No. of bitstreams: 1 Ruy_Fernando_D.pdf: 990685 bytes, checksum: 783dfc6c0515d5c6cf6b7f57ded8fcd4 (MD5) Previous issue date: 2007 / Resumo: A levedura Candida albicans é um importante patógeno em humanos. A principal característica deste microorganismo é a sua habilidade de sobreviver em diferentes ambientes do hospedeiro, com diferentes tensões de oxigênio. Acredita-se que esta flexibilidade deve-se à expressão de vias alternativas de redução do oxigênio e ao controle do fluxo de elétrons entre elas e a cadeia respiratória clássica. A presença destas vias pode ainda estar relacionada à alta resistência do fungo a drogas, uma vez que a inibição de uma delas pode ser suprida pela atividade das outras. Neste sentido, um entendimento detalhado do transporte de elétrons destas leveduras torna-se imprescindível. Neste trabalho, após a confirmação da existência de três vias de transporte de elétrons nas mitocôndrias de C. albicans: a cadeia clássica (CRC), uma via paralela (PAR) e a oxidase alternativa (AOX), avaliou-se a importância de cada uma delas para o crescimento celular. Assim, foram feitos tratamentos com compostos que possuem efeito inibitório sobre pontos específicos a cadeia respiratória da levedura, e avaliações de curvas de crescimento dos organismos. A antimicina e o KCN, que inibem os Complexos III e IV da CRC, respectivamente, preveniram totalmente o crescimento quando comparadas à cultura controle. O BHAM, um inibidor da AOX, também promoveu a inibição da proliferação celular. A presença de rotenona, um inibidor do Complexo I da CRC, não inibiu completamente o crescimento da levedura. Estes experimentos foram então correlacionados com medidas de consumo de oxigênio nas suspensões mitocondrial e celular, onde nós observamos que a rotenona e o BHAM foram capazes de promover apenas inibições parciais da respiração. As concentrações de antimicina e KCN que promoveram inibições respiratórias parciais proporcionaram efeitos distintos sobre o crescimento. Enquanto a antimicina preveniu totalmente o crescimento, o KCN não apresentou efeito inibitório. Avaliamos ainda os níveis de espécies reativas de oxigênio (EROs) e glutationa oxidada (GSSG) nas células incubadas na presença dos inibidores respiratórios. Os níveis de EROs nas mitocôndrias tratadas com antimicina foram significativamente aumentados em relação aos outros grupos. Observamos também um aumento de GSSG nas células incubadas com antimicina e BHAM. Nossas descobertas experimentais sugerem que as inibições do Complexo III da CRC e a AOX previnem o crescimento da levedura por promover estresse oxidativo de origem mitocondrial / Abstract: Candida albicans is an important pathogen in humans. A crucial feature of this microorganism is its ability to survive in different environments within the host with distinct oxygen tensions. It is believed that this flexibility is due the expression of alternative oxygen reduction pathways and the control of electron flux between them and the classical respiratory chain. The presence of these pathways may also be related to the high resistance these fungi present to drugs, since the inhibition of one electron transport pathway can be compensated by the activity of the others. In this study we confirm the presence of three electron transport pathways in Candida albicans: the classical respiratory chain (CRC), a parallel chain (PAR) and an alternative oxidase (AOX). We also evaluated the contribution of each pathway toward yeast growth. To do so, we measured cellular proliferation in the absence and presence of electron transport inhibitors. Antimycin A and KCN, which inhibit Complexes III and IV, respectively, totally prevented cellular growth. BHAM, an AOX inhibitor, also promoted inhibition of yeast growth. Surprisingly, the presence of rotenone, an inhibitor of respiratory Complex I, inhibited yeast growth only partially. These results were then correlated with oxygen uptake in mitochondrial and cell suspensions, which revealed that rotenone and BHAM promote only partial respiratory inhibitions. Concentrations of antimycin and KCN which promoted partial respiratory inhibition had distinct effects on growth. While antimycin totally prevented cell proliferation, KCN did not. We also evaluated the levels of reactive species of oxygen (ROS) and oxidized glutathione in the cells incubated in the presence of respiratory inhibitors. The levels of ROS in antimycin-treated cells were significantly higher than in the other groups. Finally, we observed an increase of oxidized glutathione in cells incubated in the presence of antimycin and BHAM. These experimental findings suggest that inhibitions of complex III of the CRC and AOX prevent Candida albicans growth by promoting oxidative stress, indicating the importance of these pathways to ensure yeast viability / Doutorado / Ciencias Biomedicas / Doutor em Ciências Médicas

Introducción. El estudio de hongos depende cada vez más de técnicas moleculares modernas como Polimerase Chain Reaction (PCR). Muchos cultivos microbianos son sometidos directamente a PCR, sin embargo, en levaduras, las técnicas para extracción de ADN son combinadas con procedimientos laboriosos y extensos para romper la pared celular. Recientemente, se ha descrito la técnica FTA ® (Flinders Technology Associated de Whatman), mediante la cual se colecta, transporta, almacena y purifica ADN en forma simultánea. El objetivo de este trabajo fue realizar un estudio comparativo de dos métodos de purificación de ADN genómico de levaduras del género Cándida, y su aplicabilidad para el análisis por la técnica de Random Amplified Polymorphic ADN mediante PCR (RAPD-PCR). Material y Método. Se comparó un método convencional basado en la ruptura de las paredes de levaduras con perlas de vidrio, combinado con extracción fenólica y el método de purificación con cartas FTA ® . Se purificó ADN genómico de 10 cepas de C. albicans, el cual fue posteriormente sometido a la técnica de RAPD-PCR, usando dos partidores diferentes. Para evaluar los resultados se analizó el conjunto de amplicones, el índice de error de amplificación (IEA), el número total de genotipos y la variabilidad genética se representó mediante un dendograma. Resultados. Para todas las cepas analizadas, con el método convencional y el partidor OPBA13 se obtuvo un único genotipo, un IEA = 0 y con el partidor CaBUO1 2 genotipos y un IEA = 0,13. En cambio, con el método FTA, usando el partidor OPBA13 se obtuvo 7 genotipos distintos, con un IEA = 0,55 y con el partidor CaBUO1 las cepas mostraron 12 genotipos y un IEA = 0,9. Conclusiones. Se observó que existen diferencias en el análisis de la variabilidad genética de levaduras del género Candida, determinada por la técnica de RAPDPCR, utilizando ADN genómico obtenido por el método convencional y por la técnica que usa las cartas FTA. Se requieren análisis adicionales para optimizar las condiciones de PCR con los filtros FTA. / Trabajo de Investigación Requisito para optar al Título de Cirujano Dentista

Zugl.: Braunschweig, Techn. Univ., Diss., 2009

Thesis (MSc)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: The ascomycetous yeast, Candida albicans, has been almost exclusively studied in a clinical context, due to the medical risk and costs associated with the yeast. Most environmental research into the external survival of this opportunistic pathogen has been concerned with short-term, severe pollution challenges. However, a study of literature indicates that the habitat characteristics of the oxygenlimited zones in wetlands and riverbanks are comparable to those of the gastrointestinal source of sewage-borne C. albicans. Interestingly, these are the external, environmental regions to which sewage-borne C. albicans is often exposed. In addition, oxygen-limitation is the predominant parameter in stimulating conjugation of C. albicans. Based on these observations, this study aimed to assess polluted river bank and wetland environments in the Western Cape of South Africa as potential habitats to accommodate a niche for C. albicans, particularly comparing the presence of this yeast in oxygen-limited, plant debris-rich zones and aerobic, clear, flowing zones. The second objective was to employ in vitro microcosm studies to investigate the survival and growth of C. albicans in various microhabitats similar to those comprising the oxygen-limited zones of wetlands. These included the rhizosphere of wetland flora, various soil and mud types and decomposing plant debris. The final objective was to establish the presence of sufficient nutrient and energy sources within this environment for the growth of C. albicans. In particular, cellulosic substrates and mono- and disaccharides released by the natural degradation of wetland plant debris were investigated as potential energy sources for this human commensal in the wetland environment. These study objectives combined to demonstrate the potential of such an oxygen-limited, plant debris-rich environment as a niche for C. albicans external to its human host. Both semi-quantitative culturing techniques and quantitative Real-Time PCR demonstrated the improved survival of C. albicans in oxygen-limited, plant debris-rich zones in wetland and river bank environments, in comparison to aerobic, clear subsurface water zones in the same environments. These zones were compared in the Plankenburg and Diep Rivers, situated in the Western Cape of South Africa. Correlations between coliform concentrations and total yeast concentrations were demonstrated in each of the different river zones, with higher pollution levels characteristic of the dry season. Candida albicans numbers in flowing water (zone W), rock-filtered (zone R) and plant-filtered water (zone P) were compared during the progress of the rainy and dry seasons. No C. albicans was observed in clear, flowing water throughout the analysis. Early in the rainy season, both rock-filtered (aerobic, poor in plant debris) and plant-filtered (oxygen-limited, rich in plant debris) water demonstrated C. albicans numbers at approximately equivalent levels of 10²-10³ cells/100 mL. However, as the rainy season progressed and total yeast and coliform numbers in all zones of the rivers dropped to negligible levels, C. albicans could no longer be detected in aerobic, rock-filtered zones; but its numbers remained at constant levels in oxygen-limited, plant-filtered zones. This suggests that oxygen-limited wetland and river bank zones rich in plant matter, analogous to the human gastrointestinal tract, may provide an ideal habitat in which C. albicans could establish a niche external to its host. The survival of this yeast in the various microhabitats that comprise this anaerobic, reducing wetland environment was evaluated with in vitro microcosms. The rhizosphere of wetland plants had no influence on C. albicans growth and survival in comparison to bulk soil away from the plant, and wetland mud microbiota was demonstrated to be inhibitory to its survival. However, decaying plant debris was shown to increase the survival of the yeast in this inhibitory mud environment. Candida albicans was shown to compete well saprophytically in anaerobic plant debris microcosms. In addition, the tendency of C. albicans to associate with plant matter in an aquatic environment was demonstrated by inoculating the yeast in water containing Hydrilla, a submerged macrophyte found in South African aquatic environments. Plate and liquid analyses, as well as an ANKOM NDF analysis, indicated unequivocally that the C. albicans strains evaluated in this work were unable to utilise the complex carbohydrates of the wetland habitat, including cellulose and fibre. However, HPLC, along with GCMS, demonstrated the anaerobic assimilation by C. albicans of monosaccharides released by natural lignocellulose degradation of wetland plant matter. An analysis of total nitrogen by digestion in a nitrogen analyser, as well as evaluation of ammonium, nitrate and nitrite in a KCL extract, also showed that C. albicans assimilates nitrogenous compounds released by the decomposition of wetland plant matter. This decay process occurs constantly in wetland and river bank habitats. It may therefore provide energy and nutrients for C. albicans, particularly in the anaerobic zones where conjugation may possibly occur and a niche may be established, as indicated by the results obtained for the Plankenburg and Diep Rivers. / AFRIKAANSE OPSOMMING: Die askomisete gis Candida albicans is feitlik eksklusief in ‘n kliniese konteks bestudeer weens die mediese risiko en koste daaraan verbonde. Die meeste omgewingsnavorsing op die eksterne oorlewing van hierdie opportunistiese patogeen was toegespits op die uitdagings van ernstige korttermyn besoedeling. ‘n Literatuurstudie toon egter dat die habitat-eienskappe van die suurstof-beperkte sones in vleilande en rivieroewers vergelykbaar is met dié van die gastroïntestinale bron van C. albicans wat in riool gevind word. Interessant genoeg is dit juis hierdie eksterne omgewingsgebiede waaraan C. albicans vanuit riool dikwels blootgestel word. Hierby is suurstof-beperking die vernaamste parameter in die stimulering van konjugasie in C. albicans. Op grond van hierdie waarnemings poog dié studie om besoedelde vleilande en rivieroewers in die Wes-Kaap Provinsie van Suid-Afrika te evalueer as potensiële habitatte wat ‘n nis van C. albicans kan akkommodeer, en veral om die teenwoordigheid van hierdie gis in suurstof-beperkte sones ryk aan plantafval te vergelyk met aerobe, helder, vloeiende sones. Die tweede doelwit was om in vitro mikrokosmos studies te gebruik om die oorlewing en groei van C. albicans in verskillende mikrohabitatte soortgelyk aan suurstof-beperkte sones in vleilande te ondersoek. Dit sluit die risosfeer van vleilandflora in, asook verskillende grond- en moddertipes en ontbindende plantafval. Die laaste doelwit was om die teenwoordigheid van genoegsame voedings- en energiebronne in dié omgewing te bepaal vir die groei van C. albicans. In besonder is sellulose substrate, asook die mono- en di-sakkariede, wat deur die natuurlike afbraak van vleiland plantafval vrygestel word, as potensiële energiebronne van hierdie mens-kommensaal in die vleiland-omgewing ondersoek. Hierdie studiedoelwitte het gesamentlik die potensiaal van so ‘n suurstofbeperkte, plantafvalryke omgewing as ‘n nis vir C. albicans buite die menslike gasheer aangetoon. Beide semi-kwantitatiewe kweektegnieke en kwantitatiewe in-tyd PKR het die verbeterde oorlewing van C. albicans in suurstofbeperkte, plantafvalryke sones in vleiland en rivieroeweromgewings gedemonstreer, in teenstelling met aerobe, helder oppervlakwatersones in dieselfde omgewings. Hierdie sones in die Plankenburg- and Dieprivier in die Wes-Kaap Provinsie, Suid-Afrika, is met mekaar vergelyk. Korrelasies tussen coliform konsentrasies en totale giskonsentrasies is in elk van die verskillende sones in dié riviere gedemonstreer, met hoër vlakke van besoedeling kenmerkend aan die droër seisoen. Candida albicans getalle in vloeiende water (sone W), rots-gefiltreerde (sone R) en plant-gefiltreerde water (sone P) is deur die verloop van die reën- en droë seisoene met mekaar vergelyk. Geen C. albicans is deur die loop van die analises in helder, vloeiende water bespeur nie. Vroeg in die reënseisoen het beide rots-gefiltreerde (aerobe, min plantafval) en plant-gefiltreerde (suurstofbeperk, ryk in plantafval) water vergelykbare vlakke van C. albicans getoon, naamlik 10²-10³ selle/100 mL. Soos die reënseisoen egter verloop het en die totale gis- en coliforme getalle in al die sones van die riviere tot weglaatbare vlakke gedaal het, kon C. albicans egter nie meer in die aerobe, rots-gefiltreerde sones bespeur word nie, hoewel die getalle in suurstofbeperkte, plant-gefiltreerde sones konstant gebly het. Dit dui daarop dat suurstof-beperkte vleiland en rivieroewer sones ryk in plantmateriaal, analoog tot die menslike gastroïntestinale kanaal, die idealke habitat mag bied waarin C. albicans ‘n nis mag vind buite sy gasheer. Die oorlewing van hierdie gis in die verskillende mikrohabitatte wat uit hierdie anaerobe, reduserende vleilandomgewing bestaan, is met in vitro mikrokosmosse geëvalueer. Die risosfeer van vleilandplante het in vergelyking met die grond weg van die plant geen effek op die groei en oorlewing van C. albicans gehad nie, en vleiland modder-mikrobiota is gevind om die oorlewing daarvan te inhibeer. Verrottende plantafval het egter die oorlewingsvlakke van giste in hierdie inhiberende modderomgewing verbeter. Candida albicans kan egter saprofities goed kompeteer in anaerobe plantafval mikrokosmosse. Hierby is die geneigdheid van C. albicans om met plantmateriaal in waterige omgewings te assosieer gedemonstreer deur die gis te innokuleer in water wat Hydrilla, ‘n onderwater makrofiet wat in Suid-Afrikaanse akwatiese omgewings aangetref word, bevat. Plaat en vloeibare analises, asook ‘n ANKOM NDF data-analise, het onteenseglik getoon dat die C. albicans stamme wat in dié werk gebruik is, nie in staat was om die komplekse koolhidrate, insluitende sellulose en vesel, van die vleiland habitat te benut nie. HPLC, saam met GC-MS, toon egter C. albicans se anaerobe assimilasie van monosakkariede wat deur natuurlike lignosellulose afbraak van vleiland plantmateriaal vrygestel is. ’n Totale stikstof analise deur vertering in ’n stikstof analiseerder, en ’n evalueering van ammonium, nitraat en nitriet in ‘n KCl ekstrak, het ook getoon dat C. albicans stikstofverbindings assimileer wat deur die afbraak van vleiland plantmatriaal vrygestel word. Hierdie afbraakproses kom deurlopend in vleiland en rivieroewer habitatte voor en verskaf potensieel energie en voedingstowwe aan C. albicans, spesifiek in die anaerobe sones waar konjugasie moontlik kan plaasvind, en ‘n nis gevestig kan word, soos aangedui deur die Plankenburg- and Dieprivier.