Made available in DSpace on 2014-07-29T15:25:24Z (GMT). No. of bitstreams: 1 CarolinaRodriguesCosta-2009.pdf: 705821 bytes, checksum: 53ed28c3e9fdc5770d04b7c412384968 (MD5) Previous issue date: 2009-06-02 / Adhesion to host tissues, production of hydrolytic enzymes, the resistance to antifungals and ability to production hyphal interfere in the infectious process caused by Candida. Resistance to azole antifungal agents, used to treatment of candidiasis, has been observed to immunocompromised patients. Molecular typing based on RAPD-PCR has been used to discriminate between susceptible and resistant isolates to antifungal agents. In this work, were evaluated the virulence factors and molecular characteristics of Candida isolates obtained of samples from blood, catheter of nosocomial patients and from oral cavity of HIV positive patients. The isolates were identified as: Candida albicans (59) Candida parapsilosis (22), Candida tropicalis (14) Candida guilliermondii (07), Candida. famata (05), Candida krusei (03), Candid. lusitaniae (01) and Candida kefyr (01). The proteinase and phospholipase production and the adherence ability were determined for these yeasts. The effect of fluconazole and itraconazole antifungal agents on hyphal formation were studied to 5 isolates previously classified as either susceptible or resistant. The characterization genotypic of resistant and susceptible isolates to fluconazole was carried out for 13 isolates of C. albicans by RAPD-PCR method. The results showed that proteinase activity was detected in 88.1% of C. albicans isolates and in 69.8% of non C. albicans, while phospholipase was produced in 55.9% of C. albicans isolates and in 37.7% of non C. albicans. Isolates of blood were more proteolitic than catheter and oral cavity, while for phospholipase, there was more production of this enzyme in the oral cavity. The ability of adherence to buccal epithelial cell was higher in C. albicans than non C. albicans, however there was not behavior difference between the isolates from different sources studied. The hyphal formation was higher in resistant isolates than susceptible isolates when used the both drugs. In RAPD-PCR method the formation of two different groups was verified for susceptible and resistant isolates being that only one resistant isolate was clustered in the susceptible group. Thus, in this work, it was verified that the exoenzymes activity and adherence ability depend not only of the specie of Candida, but too of the source from host; the resistant isolates produced more hyphal than susceptible isolates under the antifungal action and the molecular characteristics of the resistant isolates did not suggest unique DNA fingerprints did not predicting their susceptibility to fluconazole / A capacidade de aderência ao tecido do hospedeiro, a produção de exoenzimas, a resistência aos antifúngicos e a formação de hifas são fatores que podem interferir no processo infeccioso causado por Candida. Resistência aos derivados azólicos utilizados no tratamento de candidíase, tem sido observada em pacientes imunocomprometidos. Tipagem molecular como o RAPD-PCR tem sido utilizada para discriminação entre isolados de Candida spp suscetíveis e resistentes aos antifúngicos. Neste trabalho foram avaliados fatores de virulência e características moleculares de leveduras do gênero Candida isoladas de amostras do sangue, de cateter de pacientes nosocomiais e da cavidade bucal de pacientes HIV positivos. Os isolados utilizados foram identificados como: Candida albicans (59) Candida parapsilosis (22), Candida tropicalis (14) Candida guilliermondii (07), Candida famata (05), Candida krusei (03), Candida lusitaniae (01) e Candida kefyr (01). Estas leveduras foram avaliadas quanto à atividade de proteinase, fosfolipase e à sua capacidade de aderência. A ação do fluconazol e itraconazol sobre a formação hifal, foi avaliada em 5 isolados previamente classificados como suscetíveis e resistentes ao fluconazol e ao itraconazol. A caracterização genotípica de 13 isolados de C albicans resistentes e suscetíveis ao fluconazol foi realizada por meio de RAPD-PCR. Os resultados mostraram que a atividade de proteinase foi detectada em 88,1% de isolados de C. albicans e em 69,8% de Candida não albicans, enquanto que a fosfolipase foi detectada em 55,9% de isolados de C. albicans e em 37,7% de Candida não albicans. Isolados do sangue foram mais proteolíticos do que os do cateter e os da cavidade bucal, enquanto para a fosfolipase foi observado maior produção desta enzima em isolados da cavidade bucal. A capacidade de aderência à célula epitelial foi maior em C. albicans que Candida não albicans, no entanto não houve diferença de comportamento entre isolados obtidos dos diferentes locais estudados. A formação de hifas foi maior nos isolados resistentes do que nos isolados suscetíveis quando sob a ação de qualquer um dos dois fármacos. Na análise do RAPD-PCR foi verificada a formação de dois grupos distintos para os isolados suscetíveis e resistentes ao fluconazol, sendo que apenas um isolado resistente foi agrupado com os suscetíveis. Neste trabalho, foi verificado que a atividade de exoenzimas e a habilidade de aderência dependem além da espécie de Candida como também do local onde foi isolada no hospedeiro, que isolados resistentes formaram mais hifas do que os suscetíveis sob a ação de antifúngico e que as características moleculares dos isolados resistentes em mais de um padrão fingerprinting não permitiram predizer a sua suscetibilidade ao fluconazol

Made available in DSpace on 2015-04-22T22:06:41Z (GMT). No. of bitstreams: 1 Shirley.pdf: 2487120 bytes, checksum: 840010088c8c6cc034faf3bb463fd6dd (MD5) Previous issue date: 2009-03-31 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Oral candidosis is an infection disease caused mainly by Candida albicans, normal commensal yeast in oral cavity of healthy individuals. The aim of this study was determine the prevalence of different species of Candida in patients of Basic Unit of Healthy (UBS)/SEMSA in Manaus city/Amazon state/Brazil, that wearing denture or present natural teeth. This transversal study was performed by analysis of oral mucosa samples. The samples were collected of 594 patients in ten UBS s and were cultured in Petri dishes containing Sabouraud Dextrose Agar with chloramphenicol and direct analysis (Gram method). The yeasts were submitted to biochemical identification with Candifast® (International Microbio) and morphological study on Corn Meal Tween 80 Agar. C. dubliniensis identification was carried out by primer-specific amplification. The analysis of the results was made by measurements of frequencies, average, median, standard deviation and confidence limits and also was used Student s t method, ANOVA tests, Pearson chi-square and Fischer Exact test. The data were analyzed by Epi-Info software, version 3.5. In the total 594 patients taken, 436 were women and 158 were men of ten UBS-SEMSA, with the average age of 32.5 ±11.6 years old. Candida species occurred in 50% of patients. C. albicans was the most prevalent species followed by C. parapsilosis, C. tropicalis, C. krusei, C. kefyr e C. glabrata. Factors like gender, age, medical treatment and use of medication were directly associated with the presence of Candida in oral cavity. In the patients taken, 188 wearing denture, with 75% of prevalence of Candida and present a significant statistical difference (p<0.05). It was confirmed, by PCR, the identity of 113 samples for C. albicans and 28 samples were identified as C. dubliniensis. We concluded that the prevalence of species of Candida in patients wearing denture, as well as individuals with natural teeth, was the same that observed in other regions of Brazil and other countries. The data suggested the specifics oligonucleotides CdF/CdR and CaF/CdR, used in this work, were effective to confirm C. albicans species and efficient to identification of C. dubliniensis species from theses isolates. / A candidíase oral é uma doença infecciosa causada geralmente pela Candida albicans, habitante comum da cavidade bucal de pessoas sadias. O objetivo deste estudo foi determinar a prevalência das diferentes espécies de Candida em pacientes atendidos nas Unidades Básicas de Saúde/SEMSA na cidade de Manaus/AM, usuários ou não de prótese dentária. Foi realizado um estudo transversal pela análise de amostras de Candida da cavidade oral. As amostras foram coletadas de 594 pacientes atendidos em 10 UBS s e cultivadas em placas de Petri contendo Ágar Sabouraud Dextrose com clorafenicol e feito exame direto (método de Gram). As leveduras foram submetidas à identificação bioquímica utilizando-se o Kit Candifast® e estudo morfológico com microcultivo em ágar fubá com Tween 80. A identificação de C. dubliniensis foi feita com primer espécie-específico. A análise dos resultados foi realizada através de medidas de freqüências, média, mediana, desvio-padrão e intervalo de confiança e utilizados os testes t de Student, ANOVA, qui-quadrado de Pearson e exato de Fisher. Os dados foram armazenados e analisados utilizado o software Epi-Info versão 3.5. Das 594 pessoas participantes, 436 são do gênero feminino e 158 do gênero masculino, com média de idade de 32,5 ± 11,6. Culturas positivas para Candida foram observadas em 50% dos pacientes. C. albicans foi a espécie mais prevalente seguida pela C. parapsilosis, C. tropicalis, C. krusei, C. kefyr e C. glabrata. Fatores como gênero, idade, tratamento médico e uso de medicamentos foram diretamente associados com a presença de Candida na cavidade oral. Do total de pacientes, 188 usavam prótese dentária, com prevalência de Candida de 75%, com diferença estatística significante (p < 0,05). Foi confirmada por PCR a identidade de 113 amostras de C. albicans e identificados 28 isolados de C. dubliniensis. Podemos concluir que a prevalência das espécies de Candida tanto em pacientes usuários de prótese como em indivíduos com dentição natural foi semelhante a prevalência encontrada em outras regiões do Brasil e outros países. Os dados sugerem, ainda, que oligonucleotídeos específicos CdF/CdR e CaF/CdR, utilizados neste estudo, foram efetivos para confirmar isolados de C. albicans e eficientes para identificar C. dubliniensis a partir desses isolados.

Candida albicans é um fungo de grande importância médica e veterinária por ser patogênico ao homem e a outros animais de sangue quente, sendo responsável por 80 a 90% das infecções fúngicas. Por apresentar sintomas semelhantes aos de infecções bacterianas sistêmicas há uma dificuldade para seu diagnostico imediato. Essas dificuldades podem levar a atrasos da terapia antifúngica, o que contribui para as altas taxas de mortalidade associadas a essa infecção. A capacidade de alternar entre levedura e hifa (dimorfismo) é um dos atributos de virulência comum a C. albicans. Estruturas de resistência denominadas clamidoconídios são muito comuns neste patógeno, representando tipos celulares distintos que se formam em resposta a determinadas condições genéticas ou ambientais. Recentemente, diversos fármacos antifúngicos e novas estratégias terapêuticas têm entrado em uso, possibilitando ao fungo a aquisição de uma resistência aos fármacos. O uso de radiação ionizante tem sido empregado para produção de imunógenos. A eficácia da ação fungicida de uma determinada dose de radiação depende de alguns fatores, tais como, fase de seu ciclo de vida e espécie. Estes fatos determinaram a presente pesquisa, que teve como objetivo avaliar os efeitos da radiação gama (60Co) em leveduras e clamidoconídios de C. albicans com doses entre 320 e 10240 Gy. Houve a perda da capacidade reprodutiva dos clamidoconídios e das leveduras em doses de 6000 Gy. A viabilidade das leveduras irradiadas manteve-se superior a 85% e os clamidoconídios mantiveram sua viabilidade superior a 70%. A análise linfoproliverativa resultou em significância de p<0,001 para ambas as formas. Através da microscopia de transmissão e de fluorescência pode-se notar alterações citoplasmáticas e anomalias na parede celular de ambas as formas morfológicas de C. albicans. / Candida albicans is a fungus of great medical and veterinary importance to be pathogenic to humans and other warm-blooded animals, accounting for 80-90% of fungal infections. Because the symptoms are similar to those of systemic bacterial infections there is a difficulty for immediate diagnosis. These difficulties can lead to delays of antifungal therapy, which contributes to the high mortality rates associated with this infection. The ability to switch between yeast and hyphae (dimorphism) is one of the attributes of common virulence of C. albicans. Resistance structures called chlamydospores are very common in the pathogen, representing different cell types that form in response to certain genetic or environmental conditions. Recently, several antifungal agents and new therapeutic strategies have come into use, allowing the fungus to acquire a resistance to the drugs. The use of ionizing radiation has been used to produce immunogens against several parasites. The efficacy of the fungicidal action of a given dose of radiation depends on factors such as stage of its life cycle and kind. These facts determined the present study, which aimed to evaluate the effects of gamma radiation (60Co) in yeast and chlamydospores of C. albicans with 320 and 10240 Gy doses. There was damage of reproductive capacity of chlamydospores at 6000 Gy doses and the reproductive capacity of yeast at 6000 Gy.doses. The viability of irradiated yeasts remained greater than 85% and chlamydospores maintained their viability higher than 70%. The lymphoproliferative analysis resulted in significance of p<0.001 for both forms. Through microscopy and fluorescence microscopy may be noted cytoplasmic changes and abnormalities in the cell wall of both morphological forms of C. albicans.

Essa tese teve como objetivo avaliar todos os casos de candidose sistêmica por Candida albicans identificadas através de kit comercial ID 32C® (bioMérieux), diagnosticados no Laboratório de Micologia da Santa Casa de Misericórdia de Porto Alegre/RS, durante o período de 1999 a 2009, buscando identificar a prevalência de C. dubliniensis, bem como avaliar os fatores de virulência e diferença de perfil de suscetibilidade antifúngica entre os isolados clínicos. Foi realizado um levantamento clínico-epidemiológico dos casos incluídos no estudo, avaliando sexo, idade, manifestações clínicas, evolução, região proveniente do paciente, doença de base, condições predisponentes, utilização de corticóides e antibióticos e resposta ao tratamento recebido. Para a diferenciação das duas espécies utilizou-se testes fenotípicos (arranjo dos clamidosporos, teste de termotolerância, formação do tubo germinativo, crescimento em meio hipertônico e niger), molecular (espectrometria de massa) e genotípico (reação em cadeia da polimerase - PCR). Em adição, foi avaliada a eficácia do método de conservação das leveduras estocadas a -20ºC e comparamos quatro substratos (soro fresco, soro congelado, ágar e caldo Mueller-Hinton) para a prova do tubo germinativo. Determinou-se a produção da fosfolipase e proteinase em isolados incluídos no estudo. A atividade in vitro dos antifúngicos fluconazol, anfotericina B e anidulafungina frente aos isolados estudados foi determinada através da concentração inibitória mínima (CIM), a concentração fungicida mínima (CFM) e ponto de corte epidemiológico (ECV). Os casos de candidemia por C. albicans diagnosticados durante 10 anos ocorreram com maior frequência em pacientes adultos com presença de cateteres. Observamos que houve maior chance de ocorrência desta em pacientes oncológicos. O percentual de alta nos pacientes foi baixo. O método utilizado para a conservação de leveduras nesse estudo apresentou taxa de 70% de viabilidade. O ágar e o caldo Mueller-Hinton demonstraram sensibilidade de 90% e especificidade de 100%. Os isolados de C. albicans provenientes de hemocultivos apresentaram produção de fosfolipase em 78% e proteinase em 97% dos isolados. A espécie C. dubliniensis não foi identificada em isolados de hemocultivos, sendo todos os casos de candidemia por C. albicans. Os testes microcultivo em ágar fubá, espectrometria de massa, caldo niger e caldo hipertônico concordaram com o teste genotípico. Os isolados de C. albicans apresentaram maior suscetibilidade a anidulafungina, entretanto, os menores valores obtidos em 90% dos isolados (CIM90) foi pela anfotericina B. E através do ECV, os isolados poderiam ser resistentes ao fluconazol, demonstrando a importância da associação desses dois parâmetros. / The aim this tesis was to evaluate systemic candidiasis cases by Candida albicans through ID 32C® (bioMérieux), at Mycology Laboratory of the Santa Casa de Porto Alegre/RS, during 1999 to 2009, seeking to identify the C. dubliniensis prevalence, as well as evaluating the virulence factors and antifungal susceptibility profile difference of among isolates. The clinical and epidemiological survey was made through gender, age, clinical manifestations, evolution, patient's region, underlying disease, predisposing conditions, steroids and antibiotics use, and response to treatment. The phenotypic tests (tthermotolerance, germ tube, hypertonic and Niger medium), molecular (mass spectrometry) and genotypic (polymerase chain reaction – PCR) was used for two species identification. We also assessed if the mantainance of C. albicans stored at - 20ºC in a freezer with sterile distilled water was usefull.The four substrate (fresh and frozen serum, agar and broth Mueller-Hinton®) were used for germ tube formation and the phospholipase and proteinase activity were evaluated. The in vitro activity of fluconazole, amphotericin B and anidulafungin were compared through the minimum inhibitory concentration (MIC), the minimum fungicidal concentration (MFC) and epidemiological cutoff value (ECV). The candidemia cases by C. albicans for ten years occurred more frequently in adult and catheters use. We observed the more chance this occurrence in cancer patients. The survival percentage was low. The used method in the study for yeast stored had 70% of viability. The agar and broth Mueller-Hinton were 90% sensitivity and 100% specificity. The boodstream isolates of C. albicans produce virulence factors, such the germ tube production and hydrolytic enzymes (78% of phospholipase and 97% of protease) production. The C. dubliniensis was not identified in bloodstream isolates, thus all candidemia cases were by C. albicans. The mass spectrometry, cornmeal agar, Niger and hypertonic broth agreed with genotypic test. The isolates exhibited more susceptibility to anidulafungin, and 90% of them (MIC90) exhibited the lowest values against amphotericin B. Based on ECV and Pfaller classification, isolates could be resistant to fluconazole, demonstrating the importance of the combination of these parameters.

Jin, Dawei. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 83-93). / Abstracts in English and Chinese. / ABSTRACT (IN CHINESE) --- p.ii / ABSTRACT (IN ENGLISH) --- p.iv / ACKNOWLEDGEMENTS --- p.vii / CONTENTS --- p.ix / LIST OF TABLES --- p.vxiii / LIST OF FIGURES --- p.xiv / LIST OF ABBREVIATIONS --- p.xvi / Chapter CHAPTER I --- INTRODUCTION --- p.1 / Chapter 1.1 --- Biology of C. albicans --- p.2 / Chapter 1.1.1 --- Taxonomy --- p.2 / Chapter 1.1.2 --- Basic cell biology --- p.2 / Chapter 1.1.2.1 --- Cell cycle and phenotypic switch --- p.2 / Chapter 1.1.2.2 --- Cell wall --- p.3 / Chapter 1.1.3 --- "Morphological, culture and biochemical characteristics" --- p.4 / Chapter 1.1.4 --- Genomics --- p.5 / Chapter 1.1.5 --- Pathogenecity --- p.6 / Chapter 1.2 --- Catheter-related bloodstream infections (CRBSI) caused by C. albicans --- p.7 / Chapter 1.2.1 --- Intravenous catheter type --- p.7 / Chapter 1.2.2 --- Epidemiology of CRBSI caused by C. albicans --- p.8 / Chapter 1.2.3 --- Pathogenesis of intravascular catheter-related infections --- p.9 / Chapter 1.2.4 --- Diagnosis of catheter-related infections --- p.10 / Chapter 1.2.5 --- Prevention and control --- p.11 / Chapter 1.3 --- Mechanism of C. albicans adhesion to catheters --- p.12 / Chapter 1.3.1 --- The definition of microbial adhesion --- p.12 / Chapter 1.3.2 --- Relationship between microbial adhesion and biofilm formation --- p.12 / Chapter 1.4 --- Agglutinin-like sequence (ALS) gene family of C. albicans --- p.14 / Chapter 1.4.1 --- Members of ALS gene family --- p.14 / Chapter 1.4.2 --- Chromosomal location of ALS genes --- p.14 / Chapter 1.4.3 --- ALS gene organization --- p.14 / Chapter 1.4.3.1 --- Three-domain structure of ALS genes --- p.15 / Chapter 1.4.3.2 --- Characterization of ALS genes. --- p.15 / Chapter 1.4.4 --- ALS gene allelic variation --- p.17 / Chapter 1.5 --- Experimental models for catheter adhesion study of C. albicans --- p.17 / Chapter 1.5.1 --- "Static adhesion model for C, albicans" --- p.18 / Chapter 1.5.1.1 --- Advantage of static adhesion model --- p.19 / Chapter 1.5.1.2 --- Limitation of static adhesion model --- p.19 / Chapter 1.5.2 --- Dynamic adhesion model for C. albicans --- p.19 / Chapter 1.5.2.1 --- Advantage of dynamic adhesion model --- p.20 / Chapter 1.5.2.2 --- Limitation of dynamic adhesion model --- p.20 / Chapter 1.5.3 --- Quantification methods of adherent cells --- p.21 / Chapter 1.5.4 --- ALS gene expression study in the in vitro model --- p.22 / Chapter 1.6 --- Aim of study --- p.22 / Chapter CHAPTER II --- MATERIALS & METHODS --- p.24 / Chapter 2.1 --- Strains used in this study --- p.25 / Chapter 2.2 --- Design of an in vitro dynamic adhesion model for C. albicans --- p.26 / Chapter 2.2.1 --- Flask --- p.26 / Chapter 2.2.2 --- Peristaltic pump --- p.26 / Chapter 2.2.3 --- Glass tube and vascular catheters. --- p.27 / Chapter 2.2.4 --- Sterility check of in vitro dynamic adhesion model --- p.27 / Chapter 2.3 --- Construction of C. albicans growth curve --- p.27 / Chapter 2.4 --- Measurement of C. albicans adhesion to catheters --- p.29 / Chapter 2.5 --- Detection of C. albicans ALS genes --- p.30 / Chapter 2.5.1 --- DNA extraction of C. albicans --- p.30 / Chapter 2.5.2 --- ALS primers design --- p.31 / Chapter 2.5.3 --- PCR reaction --- p.32 / Chapter 2.5.4 --- Gel electrophoresis --- p.32 / Chapter 2.5.5 --- Purification of PCR products --- p.33 / Chapter 2.6 --- Construction of E. coli plasmid containing gene --- p.34 / Chapter 2.6.1 --- Ligation using the pGEM®-T Easy Vector --- p.34 / Chapter 2.6.2 --- Preparation of E. coli DH5a electro-competent cells --- p.35 / Chapter 2.6.3 --- Clean up of DNA ligation reaction for electro-transformation --- p.36 / Chapter 2.6.4 --- Electro-transformation of E. coli DH5a electro-competent cells --- p.37 / Chapter 2.6.5 --- Blue / white screening for positive transformation of E. coli DH5a. --- p.37 / Chapter 2.6.6 --- Extraction of plasmid containing ALS1 gene --- p.39 / Chapter 2.6.7 --- Plasmid validation by PCR and gel electrophoresis --- p.39 / Chapter 2.6.8 --- Serial dilution of plasmid solutions for ALS1 standard curve construction --- p.40 / Chapter 2.7 --- C. albicans ALS1 gene expression in dynamic adhesion model --- p.41 / Chapter 2.7.1 --- Design of real-time PCR primers specific for C. albicans ALS1 --- p.41 / Chapter 2.7.2 --- Validation of primers specificity --- p.42 / Chapter 2.7.3 --- RNA extraction of C. albicans cells adhered on catheters --- p.43 / Chapter 2.7.4 --- Complementary DNA (cDNA) synthesis --- p.45 / Chapter 2.7.5 --- Quantitative real-time RT-PCR --- p.46 / Chapter 2.8 --- Statistical analyses --- p.48 / Chapter CHAPTER III --- RESULTS --- p.49 / Chapter 3.1. --- Validation of the in vitro dynamic adhesion model for C. albicans --- p.50 / Chapter 3.2. --- C. albicans growth curve construction --- p.50 / Chapter 3.3. --- Measurement of C. albicans adhesion on catheters --- p.50 / Chapter 3.4. --- Detection of C. albicans SC5314 ALS genes --- p.52 / Chapter 3.5. --- Validation of E. coli plasmid containing ALS1 gene --- p.54 / Chapter 3.6. --- C. albicans ALS 1 gene expression in dynamic adhesion model --- p.54 / Chapter 3.6.1. --- Specificity validation of ALS1 real-time primers --- p.55 / Chapter 3.6.2. --- Quantitative real-time RT-PCR --- p.55 / Chapter CHAPTER IV --- DISCUSSION --- p.57 / Chapter 4.1 --- Experimental design of the in vitro dynamic adhesion model --- p.58 / Chapter 4.1.1 --- Advantages of this in vitro dynamic adhesion model --- p.58 / Chapter 4.1.2 --- Limitation of this in vitro dynamic adhesion model --- p.58 / Chapter 4.1.3 --- Catheter arrangement inside the glass tube --- p.60 / Chapter 4.1.4 --- Reproducibility of experiments in the model --- p.62 / Chapter 4.1.5 --- Identification of potential contamination in the model --- p.63 / Chapter 4.1.6 --- Advantages of removing method for C. albicans adherent cells --- p.64 / Chapter 4.1.7 --- Limitation of removing method for C. albicans adherent cells --- p.64 / Chapter 4.1.8 --- Limitation of statistical analysis --- p.66 / Chapter 4.1.9 --- Primers design --- p.67 / Chapter 4.1.9.1 --- Primers of C. albicans ALS gene detection --- p.67 / Chapter 4.1.9.2 --- Validation of ALS 1 real-time primers specificity --- p.69 / Chapter 4.2 --- C. albicans adhesion to catheters --- p.70 / Chapter 4.2.1 --- Theoretical explanation of C. albicans adhesion to different catheters --- p.71 / Chapter 4.3 --- C. albicans ALS gene expression --- p.74 / Chapter 4.3.1 --- Functions of Als proteins --- p.75 / Chapter 4.3.1.1 --- Adhesive functions --- p.75 / Chapter 4.3.1.2 --- Other functions in C. albicans pathogenesis --- p.75 / Chapter 4.3.2 --- Analysis of ALS1 gene expression pattern in the in vitro model --- p.76 / Chapter 4.4 --- Clinical application of our study --- p.78 / Chapter 4.5 --- Future study --- p.80 / Chapter 4.6 --- Conclusion --- p.81 / REFERENCES --- p.83

A APECED é uma doença que apresenta autoimunidade e susceptibilidade a Candida albicans. Nosso grupo observou que a proteína AIRE participa da via da Dectina-1, importante contra a C. albicans. Neste projeto, investigamos como a ausência de AIRE influencia em eventos para a eliminação do patógeno via Dectina-1. Assim, avaliamos o burst oxidativo, expressão de moléculas do sistema NADPH oxidase e fagocitose por células de paciente com APECED e células THP-1 silenciadas para AIRE. Não houve diferença no burst oxidativo e na expressão dos componentes do sistema NADPH oxidase por estas células e as células silenciadas fagocitam menos que as células selvagens. Observamos que não há diferença na expressão flavocitocromo b558 e p40phox do paciente comparado ao controle. Em paralelo, mostramos que as células do paciente apresentaram um burst oxidativo e fagocitose diminuídos comparado ao controle. Estes resultados sugerem que há um defeito no reconhecimento via Dectina-1, gerando uma diminuição da fagocitose que pode dificultar sua eliminação. / The APECED is a syndrome with autoantibodies and Candida albicans susceptibility. Our group has noted that AIRE protein is required for dectin-1 signaling, important against C. albicans. In this project, we investigate how the absence of AIRE influences in events for elimination of pathogen via dectin-1. We evaluated reactive oxygen species production, expression of NADPH oxidase molecules and phagocytosis by APECED pacient cells or AIRE silent THP-1 cells. We didnt observe differences in oxidative burst and expression of NADPH oxidase components by these cells and silent cells phagocytize less than wild-type cells. We observed no difference in flavocytochrome b558 and p40phox expression in paciente cells and control. In parallel, we showed that pacient cells has a decrease in burst oxidative and phagocytosis compared to control. Our results suggest that there is a defect in pathogen recognition via dectin-1, resulting in decrease on phagocytosis that can hamper their elimination.

Orientador: Marco Antonio Bottino / Banca: Laís Regiane da Silva Concilio / Banca: Alexandre Luiz Souto Borges / Resumo: O objetivo deste estudo foi avaliar a topografia e a formação de biofilme na superfície de cerâmicas feldspáticas obtidas através de duas técnicas de confecção e dois tratamentos de superfície, assim como avaliar a viabilidade do crescimento de fibroblastos gengivais humanos (FMM-1) sobre estes materiais. Foram confeccionados 52 blocos de cada tipo de cerâmica feldspática: VM9 obtida através da técnica da estratificação e cerâmica Vita Blocs Mark II (VMII) para o sistema CAD/CAM (ambas, Vita Zahnfabrik). As superfícies dos blocos foram padronizadas em politriz nas dimensões de 4,5 x 4,5 x 1,5 mm e os blocos foram divididos em dois tratamentos de finalização de superfície: polimento com borrachas Ceramisté + pasta de polimento e aplicação de glaze spray + sinterização. Os parâmetros de rugosidade Ra e Rsm foram mensurados através de um rugosímetro de contato. As amostras foram esterilizadas e, em seguida contaminadas (n=10) para formação de biofilme heterotípico inicial de S. mutans, S. sanguinis e C. albicans, cuja aderência foi quantificada por contagem de unidades formadoras de colônias (UFC/mL). O teste MTT foi empregado para avaliação da viabilidade celular dos materiais ao crescimento de fibroblastos gengivais humanos (FMM-1) em 24 h e 7 dias (n=12). Foram realizadas análises qualitativas da superfície dos espécimes através de microscopia eletrônica de varredura (MEV) e perfilometria óptica 3D. A energia livre de superfície (ELS) foi calculada a partir de análises de gon... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract:The Objective of this study was to evaluate the topography and surface biofilm formation of feldspathic ceramics obtained through two techniques of preparation and two surface treatments, as well as to evaluate the viability of human gingival fibroblasts (FMM-1) growth on these materials. A total of 52 blocks of each type of feldspathic ceramic were made: VM9 obtained by the stratification technique and Vita Blocs Mark II (VMII) for the CAD/CAM system (both, Vita Zahnfabrik). The blocks' surfaces were standardized in a polishing machine to the dimensions of 4.5 x 4.5 x 1.5 mm and blocks were divided into two surface finishing treatments: polishing with Ceramisté rubbers + polishing with paste; and glaze application + sintering. The Ra and RSm roughness parameters were measured through a contact rugosimeter. Samples were sterilized and then contaminated (n = 10) for initial heterotypic biofilm formation of S. mutans, S. sanguinis and C. albicans, whose adherence was quantified by counting colony forming units (CFU/mL). The MTT test was used to evaluate the cellular viability of the materials to the growth of human gingival fibroblasts (FMM-1) in 24 h and 7 days (n=12). Qualitative analyzes of the specimens' surface were performed using a scanning electron microscopy (SEM) and 3D optical profilometry. Surface free energy (SFE) was calculated from goniometry analyzes of polar and apolar liquids in 10 samples of 15 x 15 x 1.5 mm. The results of Ra, RSm and ELS were subjected to 2-way ANOVA (Material x surface treatment) followed by Tukey's test (both, α=95%), and UFC (material x surface treatment x microorganisms) and MTT (material x surfacetreatment x time) data were evaluated by 3-way ANOVA and Tukey's test (α=95%). SEM and profilometry images were described. The polished ceramics presented lower roughness (Ra p=0.015; RSm p=0.049) ... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

Thesis--University of Turku, Finland, 2003. / Includes bibliographical references.

Thesis--University of Turku, Finland, 2003. / Includes bibliographical references.

Essa tese teve como objetivo avaliar todos os casos de candidose sistêmica por Candida albicans identificadas através de kit comercial ID 32C® (bioMérieux), diagnosticados no Laboratório de Micologia da Santa Casa de Misericórdia de Porto Alegre/RS, durante o período de 1999 a 2009, buscando identificar a prevalência de C. dubliniensis, bem como avaliar os fatores de virulência e diferença de perfil de suscetibilidade antifúngica entre os isolados clínicos. Foi realizado um levantamento clínico-epidemiológico dos casos incluídos no estudo, avaliando sexo, idade, manifestações clínicas, evolução, região proveniente do paciente, doença de base, condições predisponentes, utilização de corticóides e antibióticos e resposta ao tratamento recebido. Para a diferenciação das duas espécies utilizou-se testes fenotípicos (arranjo dos clamidosporos, teste de termotolerância, formação do tubo germinativo, crescimento em meio hipertônico e niger), molecular (espectrometria de massa) e genotípico (reação em cadeia da polimerase - PCR). Em adição, foi avaliada a eficácia do método de conservação das leveduras estocadas a -20ºC e comparamos quatro substratos (soro fresco, soro congelado, ágar e caldo Mueller-Hinton) para a prova do tubo germinativo. Determinou-se a produção da fosfolipase e proteinase em isolados incluídos no estudo. A atividade in vitro dos antifúngicos fluconazol, anfotericina B e anidulafungina frente aos isolados estudados foi determinada através da concentração inibitória mínima (CIM), a concentração fungicida mínima (CFM) e ponto de corte epidemiológico (ECV). Os casos de candidemia por C. albicans diagnosticados durante 10 anos ocorreram com maior frequência em pacientes adultos com presença de cateteres. Observamos que houve maior chance de ocorrência desta em pacientes oncológicos. O percentual de alta nos pacientes foi baixo. O método utilizado para a conservação de leveduras nesse estudo apresentou taxa de 70% de viabilidade. O ágar e o caldo Mueller-Hinton demonstraram sensibilidade de 90% e especificidade de 100%. Os isolados de C. albicans provenientes de hemocultivos apresentaram produção de fosfolipase em 78% e proteinase em 97% dos isolados. A espécie C. dubliniensis não foi identificada em isolados de hemocultivos, sendo todos os casos de candidemia por C. albicans. Os testes microcultivo em ágar fubá, espectrometria de massa, caldo niger e caldo hipertônico concordaram com o teste genotípico. Os isolados de C. albicans apresentaram maior suscetibilidade a anidulafungina, entretanto, os menores valores obtidos em 90% dos isolados (CIM90) foi pela anfotericina B. E através do ECV, os isolados poderiam ser resistentes ao fluconazol, demonstrando a importância da associação desses dois parâmetros. / The aim this tesis was to evaluate systemic candidiasis cases by Candida albicans through ID 32C® (bioMérieux), at Mycology Laboratory of the Santa Casa de Porto Alegre/RS, during 1999 to 2009, seeking to identify the C. dubliniensis prevalence, as well as evaluating the virulence factors and antifungal susceptibility profile difference of among isolates. The clinical and epidemiological survey was made through gender, age, clinical manifestations, evolution, patient's region, underlying disease, predisposing conditions, steroids and antibiotics use, and response to treatment. The phenotypic tests (tthermotolerance, germ tube, hypertonic and Niger medium), molecular (mass spectrometry) and genotypic (polymerase chain reaction – PCR) was used for two species identification. We also assessed if the mantainance of C. albicans stored at - 20ºC in a freezer with sterile distilled water was usefull.The four substrate (fresh and frozen serum, agar and broth Mueller-Hinton®) were used for germ tube formation and the phospholipase and proteinase activity were evaluated. The in vitro activity of fluconazole, amphotericin B and anidulafungin were compared through the minimum inhibitory concentration (MIC), the minimum fungicidal concentration (MFC) and epidemiological cutoff value (ECV). The candidemia cases by C. albicans for ten years occurred more frequently in adult and catheters use. We observed the more chance this occurrence in cancer patients. The survival percentage was low. The used method in the study for yeast stored had 70% of viability. The agar and broth Mueller-Hinton were 90% sensitivity and 100% specificity. The boodstream isolates of C. albicans produce virulence factors, such the germ tube production and hydrolytic enzymes (78% of phospholipase and 97% of protease) production. The C. dubliniensis was not identified in bloodstream isolates, thus all candidemia cases were by C. albicans. The mass spectrometry, cornmeal agar, Niger and hypertonic broth agreed with genotypic test. The isolates exhibited more susceptibility to anidulafungin, and 90% of them (MIC90) exhibited the lowest values against amphotericin B. Based on ECV and Pfaller classification, isolates could be resistant to fluconazole, demonstrating the importance of the combination of these parameters.