Glucans are (1→3)-β-linked linear and branched polymers containing anhydroglucose repeat units. They comprise a major portion of the cell wall of saprophytic and pathogenic fungi. Glucans activate a wide range of innate immune responses. They are also released from the fungal cell wall as exopolymers into the blood of patients with fungal infections. Extensive studies have been done on glucans isolated from saprophytic fungi, such as Saccharomyces cerevisiae; however, much less is known about the glucans produced by the polymorphic fungal pathogen Candida albicans. We have undertaken an extensive structural characterization and comparison of glucans isolated from C. albicans blastospores and hyphae using high-resolution, solution-state proton nuclear magnetic resonance spectroscopy (NMR). In addition, we developed a simple and straightforward method for the production of Candida hyphae that resulted in gram quantities of hyphal mass. Also, we compared and contrasted the Candida glucans isolated by two different protocols with those isolated from S. cerevisiae. Isolation protocols provide high purity glucans with source-based structural differences. Structural details provided by this NMR analysis included the degree of polymerization, molecular weight, degree and type of branching, and structural composition. We observed that Candida glucans, derived from blastospores or hyphae, are different compared to those isolated from S. cerevisiae with regard to side-chain branching along the backbone and at the reducing terminus. These structural details are an important prerequisite for biomedical studies on the interaction of isolated fungal cell wall glucans with the innate immune system.

Mycorrhizal fungi are a major component of agricultural systems and play a key role in plant nutrition. Little is known about the effects of tillage practices and manuring on arbuscular mycorrhizal fungi (AMF). The purpose of this study was to evaluate the effect of soil disturbance on winter survival, development and distribution of AMF in soil and on plant nutrient uptake and productivity. This research was conducted in long-term corn plots in two soils and under controlled conditions. / A growth chamber study with field soil demonstrated that most of the fungal hyphae with mycorrhizal plants were mycorrhizal rather than saprophytic. This result was extrapolated to subsequent experiments. Soil disturbance reduced corn nutrient uptake and growth by disrupting the AMF hyphal network. Similarly, fallow periods reduced density of AMF hyphae, leading to reduced mineral nutrients uptake and plant growth. Soil disturbance was also found to severely reduce winter survival of AMF hyphae in agricultural soil. AM hyphae could survive the winter in soil, even when they were not attached to roots. Their survival however, was improved when they remained attached to roots. / Under field conditions, indigenous AMF were more abundant in no-till soil, less abundant under reduced tillage and least abundant under conventional tillage. Under all tillage systems, most of AMF hyphae were located in the top 15 cm of the soil profile suggesting that deep plowing could result in dilution of AMF propagules in the seeding zone. There was a seasonal variation in the abundance of hyphae in soil. Soil hyphae and root colonization declined after the silking stage of corn. Hyphal abundance decreased further over the winter, to reach their lowest level in the spring. / The spatial distribution of fungal hyphae in the field was not homogenous. Hyphal density was maximal directly under the corn rows and decreased linearly up to the mid-row. Marked seasonal variations in hyphal densities were observed on the row but fluctuations at mid-row were not significant suggesting that little AMF hyphae were ever present between the rows. Liquid dairy manure had little effect on the abundance of hyphae and spores.

No description available.

Magister Chirurgiae Dentium - MChD (Oral Medicine and Periodontics) / Candida species are frequently isolated from oral mucosal surfaces of healthy individuals and is the most common genus responsible for up to 75% of all candidal infections. The most common problems associated management of oral candidiasis are antifungal drug resistance and side effects Natural medicine is an emerging field and is being explored to overcome drug resistance and to reduce side effects. Gymnemagenin (will be known as Gymnemic acid; GA) is a purified extract from Gymnema sylvestre, a slow growing, perennial, medicinal plant found in Central and Western India, Tropical Africa and Australia is regarded as one of the plants with potent antimicrobial and antifungal activity.

Orientador: Angélica Zaninelli Schreiber / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-25T22:10:52Z (GMT). No. of bitstreams: 1 SilvaFonseca_AdenilzaCristinada_M.pdf: 2857134 bytes, checksum: 9329779ba094b04efc1a51c3932a5dfd (MD5) Previous issue date: 2014 / Resumo: Introdução: Mucormicose é uma infecção oportunista invasiva causada por fungos filamentosos denominados de Mucorales, antes Zygomycetes, de difícil tratamento e, com mau prognóstico em pacientes imunocomprometidos, caracterizada por manifestações rinocerebrais, pulmonares ou disseminadas. Espécies dos gêneros Rhizopus, Mucor, Absidia (Lichtheimia) e Rhizomucor, são os Mucorales mais isolados de pacientes. São micro-organismos resistentes a voriconazol e equinocandinas in vitro e in vivo. A terapia inclui a reversão dos fatores predisponentes, retirada cirúrgica da área infectada e administração de antifúngicos, em geral, anfotericina B, de atividade terapêutica limitada e muitos efeitos colaterais. Objetivos: Identificação dos isolados de Mucorales selecionados para o estudo por metodologia clássica e técnicas moleculares; padronização da técnica de microdiluição em caldo para avaliação de suscetibilidade aos antifúngicos isolados: anfotericina B; 5-fluorocitosina; fluconazol; micafungina; itraconazol; voriconazol; miconazol e terbinafina de outros gêneros de Mucorales que não Rhizopus spp.; avaliação da Concentração Inibitória Fracional para determinação do tipo de interação que ocorre entre as combinações de antifúngicos anfotericina B x itraconazol; anfotericina B x voriconazol; terbinafina x itraconazol; terbinafina x voriconazol e terbinafina x anfotericina B; padronização da metodologia de avaliação dinâmica de crescimento para o gênero Rhizopus sp. e Rhizopus oryzae (LIF 1832), para possível estabelecimento de correlação clínico-laboratorial já que este foi isolado de um paciente em tratamento de mucormicose. Metodologia: Estudo realizado com 10 isolados clínicos de Mucorales com identificação morfológica prévia de gênero. A identificação molecular foi realizada com os iniciadores ITS1/ITS4; ITS4/ITS5 e NL1/NL4. Os testes de suscetibilidade aos antifúngicos isolados, in vitro, foram realizados pelo método de microdiluição em caldo (CLSI M38-A2). Os antifúngicos combinados foram analisados de acordo com a metodologia do "tabuleiro de xadrez". Já a avaliação dinâmica de crescimento de hifas frente aos antifúngicos anfotericina B, itraconazol e terbinafina para o isolado Rhizopus oryzae (LIF 1832) foi realizada através do sistema Biocell-Tracer?. Resultados: As identificações morfológica e molecular foram discordantes para os isolados LIF 1237 e 1832, classificados morfologicamente como Absidia (Lichtheimia) sp. e Rhizomucor sp. e identificados como Rhizopus oryzae após sequenciamento de DNA. Considerando todos os isolados, as concentrações inibitórias mínimas (CIMs) foram de: 8 a ? 16 µg/mL para micafungina, de 0,25 a 8 µg/mL para anfotericina B, ? 64 µg/mL para 5-fluorocitosina, de 16 a ? 64 µg/mL para fluconazol, de 1 a > 8 µg/mL para itraconazol, > 8 µg/mL para voriconazol; de 0,25 a 4 µg/mL para miconazol e de 0,031 a > 128 µg/mL para terbinafina. As interações resultantes da combinação de antifúngicos foram de: 100% de sinergismo entre anfotericina B x voriconazol e anfotericina B x itraconazol; 90% de sinergismo entre terbinafina x itraconazol; 80% de sinergismo entre terbinafina x voriconazol e 100% de sinergismo para a combinação de terbinafina x anfotericina B. As taxas de inibição de crescimento das hifas do isolado Rhizopus oryzae (LIF 1832) frente aos valores de CIMs obtidos pelo método de microdiluição em caldo foram: 88.25% para anfotericina B; 0% para itraconazol e 98,70% para terbinafina. O teste com a combinação de concentrações séricas de itraconazol e terbinafina resultaram em 49,8% de taxa de inibição. Conclusões: Foi observada divergência entre identificação morfológica e molecular para dois isolados. Perfis diferentes de sensibilidade aos antifúngicos foram observados dependendo das espécies. Os antifúngicos mais ativos contra a maioria dos isolados foram anfotericina B e itraconazol. Valores de CIM bem baixos para terbinafina foram observados para Cunninghamella bertholletiae e Syncephalastrum racemosum e todos os isolados foram altamente resistentes a micafungina, 5-fluorocitosina, fluconazol e voriconazol. Os testes de antifúngicos combinados mostraram redução da CIM dos antifúngicos em associação. CIMs elevadas para terbinafina foram observadas para os isolados de R. oryzae e R. stolonifer enquanto que no teste de combinação valores baixos de CIMs foram encontrados, o que ressalta a importância da combinação de antifúngicos no manejo de mucormicose. A taxa de inibição do crescimento das hifas para o isolado LIF 1832 frente aos antifúngicos isolados sugere que hifas podem ser mais suscetíveis que esporos frente aos antifúngicos avaliados. Apesar de não muito alta, a taxa de inibição de crescimento das hifas frente à anfotericina B e à combinação das concentrações séricas de terbinafina e itraconazol pode estar favoravelmente relacionada ao desfecho clínico do paciente em tratamento de mucormicose / Abstract: Introduction: Mucormycosis is an invasive opportunistic infection caused by filamentous fungi called Mucorales (formerly Zygomycetes), difficult to treat, and with poor prognosis in immunocompromised patients, characterized by rhino-cerebral, pulmonary or disseminated manifestations. Species of the genera Rhizopus, Mucor, Absidia (Lichtheimia) and Rhizomucor are the Mucorales most commonly isolated from patients and show resistance to voriconazole and echinocandins in vitro and in vivo. Therapy includes to reverse predisposing factors, surgical removal of the infected area and administration of antifungal agents, in general, Amphotericin B, with limited therapeutic activity and many side effects. Objectives: identification of selected Mucorales isolates by morphological and molecular methods; standardization of broth microdilution for antifungal susceptibility assessment of other Mucorales genera except Rhizopus to the alone antifungals: amphotericin B, fluorocytosine, fluconazole, Itraconazole, voriconazole ; miconazole and terbinafine; establish Fractional Inhibitory Concentration to determine the type of interaction that occurs between combinations of Amphotericin B/Itraconazole; Amphotericin B/Voriconazole; Terbinafine/Itraconazole; Terbinafine/Voriconazole and Terbinafine/Amphotericin B; standardization of the dynamic growth methodology for evaluation Rhizopus sp. and study of isolate Rhizopus oryzae (LIF 1832), for possible establishment of clinical-laboratory correlation this was isolated from a patient undergoing treatment for mucormycosis. Material and Methods: This study evaluated 10 clinical Mucorales isolates with previous morphological gender identification. The molecular identification was performed with primers ITS1/ITS4; ITS4/ITS5 and NL1/NL4. The antifungal susceptibility in vitro was performed by broth microdilution method (CLSI M38-A2). Antifungal combinations were analyzed according to the "chessboard" methodology . The dynamic evaluation of hyphal growth for the isolate Rhizopus oryzae (LIF 1832) for the antifungals amphotericin B, terbinafine and itraconazole, was carried through the BioCell-Tracer system. Results: The morphological and molecular identifications were discordant for LIF 1237 and LIF 1832 isolates, classified morphologically as Absidia (Lichtheimia) sp. and Rhizomucor sp. and after DNA sequencing both as Rhizopus oryzae. Considering all isolates, the minimum inhibitory concentrations (MICs) were 8 to ? 16 µg/mL for Micafungin; from 0.25 to 8 µg/mL for Amphotericin B , ? 64 µg/mL for 5 ¿ Fluorocytosine; of 16 to ? 64 µg/mL for Fluconazole; 1 to > 8 µg/mL for Itraconazole; > 8 µg/mL for Voriconazole ; 0.25 to 4 µg/mL for Miconazole and from 0.031 to > 128 µg/mL for Terbinafine. Interactions resulting from the combination of antifungals were: 100% synergism for Amphotericin B/Voriconazole and Amphotericin B/Itraconazole; 90% synergism between Terbinafine/Itraconazole; 80% synergism between Terbinafine/Voriconazole and 100% synergism for the combination Terbinafine/ Amphotericin B. The hyphae growth inhibition rates obtained for microdilution method MIC values for Rhizopus oryzae (LIF 1832) were 88.25%, for Amphotericin B; 0% for itraconazole and 98.70% to terbinafine. Combined Itraconazole and Terbinafine serum concentrations showed 49.8% of growth inhibition rate. Conclusions: Divergence between morphological and molecular identification for two isolates was observed. Different antifungal susceptibility profiles were observed depending on the species. The most active antifungal agents were Amphotericin B and Itraconazole. MICs for Terbinafine very low were observed to the Syncephalastrum racemosum and C.bertholletiae and all isolates were highly resistant to Micafungin, 5- Fluorocytosine, Fluconazole and Voriconazole. It was possible to observe antifungal MIC reduction in combined tests. MICs high for terbinafine alone were observerd to the Rhizopus oryzae and Rhizopus stolonifer while in the combo test low MIC values were observed, which highlights the importance of combining antifungal drugs in the management of mucormycosis. The hypha growth inhibition rates obtained for Rhizopus oryzae (LIF 1832) against antifungal isolates suggest that hyphae may be more susceptible to antifungal agents against spores. Although not too high, the hyphae growth inhibition rate obtained for the combination serum achievable concentrations of Terbinafine and Itraconazole can probably be related to the clinical outcome / Mestrado / Ciencias Biomedicas / Mestra em Ciências Médicas

No description available.

The ability of Candida albicans to change morphology plays several roles in its virulence and as a human commensal. The yeast-to-hyphae transition is tightly regulated by several sets of activating and repressing pathways. The DNA-binding proteins Rfg1p, Nrg1p and the global repressor Tup1p are part of the repressors found to regulate this morphogenesis. Knowledge of these repressors is based on extrapolations from homology to S. cerevisiae and from expression studies of mutants in inducing conditions, all of which are indirect means of determining a protein's function. We proposed a genome-wide location study of the Nrg1 and Rfg1 transcription factors to obtain direct data to identify their in vivo targets. Our results suggest different avenues for Nrg1p function and a regulation behaviour diverging from the previously suggested model: Nrg1p acts not only as a repressor but also as a transcription activator. Furthermore it regulates its target genes through binding in their coding regions instead binding to the expected regulatory elements on promoters.

No description available.

Candida albicans is an opportunistic pathogen that is present in the normal flora in a majority of individuals. One key factor in C. albicans virulence is the ability to change its morphology from yeast to an elongated or hyphal form. The regulation of this morphogenesis relies in part upon quorum sensing (QS) molecules. C. albicans often exists as part of a mixed culture alongside other microbes and is influenced by their presence as well as the presence of QS molecules that they produce. In this study, a library of diploid homozygous transcriptional regulator knockout (TRKO) mutants were screened to identify strains capable of forming hyphae in the presence of Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli. We identified three strains that showed increased hyphae development compared to wild type C. albicans. The strains identified had deletions of the transcriptional regulating genes Orf19.3928, Orf19.2842 (GZF3), and Orf19.3865 (RFX1). These strains were tested for alterations of filamentation in liquid media, and biofilm formation. All three strains showed increased rates of biofilm formation compared to the wild type. Orf19.3928 showed altered response to farnesol, a marked in biofilm formation and no inhibition of filamentation when farnesol was present in liquid media. The GZF3 deletion strain showed enhanced filamentation with all three bacterial species while the RFX1 deletion strain showed increased filamentation only with E. coli and S. aureus. In spent media, GZF3 showed slight increases in filamentation in E. coli and S. aureus while RFX1 had moderate increases in filamentation in E. coli and S. aureus and slight increases with P. aeruginosa.

No description available.