No description available.

Candida albicans belongs to serious human opportunistic pathogens, causing severe health complications to immunocompromised patients. To my best knowledge, it is the only organism that survives with unmethylated cap structures found on the 5'ends of mRNA molecules. Using functional assay, I demonstrated that orf19.7626 codes for C. albicans translation initiation factor 4E (Ca4E). We couldn't prove our hypothesis, that Ca4E could be responsible for the unmethylated cap recognition in our model organism S. cerevisiae. Candida sp. possesses also another rather unusual feature - ambiguous CUG codon. In most of the cases, CUG is decoded as a serine, but sometimes also as a leucine. This gives rise to a so called "statistical proteome". One CUG codon is also part of the mRNA coding for Ca4E protein, therefore two versions of Ca4E-Ca4ELeu and Ca4ESer -might occur in C. albicans simultaneously. Both of them are able to rescue deletion of S. cerevisiae eIF4E gene, but they confer temperature sensitivity to the heterologous host. This phenotype is more pronounced with the Ca4ELeu version. We observed milder temperature sensitive phenotype after co-expression of Ca4E together with C. albicans eIF4G (Ca4G). Conformational coupling between eIF4E and eIF4G leads to enhanced affinity of eIF4E to the cap...

Fungal infections are caused by a variety of fungi, and with a variety of clinical manifestations. Antifungal treatments are limited due to host toxicity and fungi gaining resistance. By utilizing the model organism Saccharomyces cerevisiae, we hope to elucidate the molecular mechanisms of fungal pathogenesis that we can then validate in the human pathogen Candida albicans, as well as explore options for novel therapies. Small molecule signaling is a method by which single-cell organisms can communicate with one another, enabling them to coordinate gene expression. This is a useful tool because it allows microbes to turn on phenotypes that are only valuable when done in large numbers, such as bioluminescence, or virulence traits. We have previously shown that the yeast Saccharomyces cerevisiae synthesizes the secondary metabolite indole-3-acetic acid (IAA) from tryptophan. IAA is secreted into the environment, where it acts as a signal. At low concentrations, the IAA signals yeast to induce virulence traits, while at high concentrations, it is lethal. The purpose of this thesis was to investigate the molecular mechanism of IAA (plant hormone auxin) regulation in fungi, specifically, Saccharomyces cerevisiae and the human pathogen Candida albicans. Towards this end, I first focused my efforts on evaluating the role of S. cerevisiae Grr1, as a putative IAA receptor. By evaluating the IAA response of several Grr1 mutants, I was able to show that the leucine-rich repeat region, while not required for function, likely plays a significant role in maintaining the structural integrity of the protein. Next, I evaluated IAA associated phenotypes, such as filamentation, surface adhesion and IAA uptake of the grr1 null mutant in the human pathogen Candida albicans. Together, these data support the hypothesis that GRR1 regulates IAA response, probably by regulating the IAA uptake carriers.

C. albicans is a polymorphic fungal pathogen which has the ability to shift from yeast to hyphae. C. albicans cell wall is composed of glucan, chitin, mannoprotein and mannan. It is not possible, using standard extraction methods, to isolate mannan from C. albicans hyphae. To isolate hyphal mannan, we developed a simplified alkali extraction method. Using this method it was determined that hyphal mannan has a much lower molecular weight, a smaller polymer distribution and altered conformation structure when compared to yeast mannan. The hyphal mannan was found to contain little to no acid-labile portion with only α-Man-PO4 groups and no long chains of β-1, 2-linked mannosyl repeat units, when compared to the yeast mannan. It was concluded that the C. albicans hyphal mannan is substantially different from the mannan found in the yeast form. This is an entirely new observation that extends the existing knowledge about the structural biology of C. albicans hyphae and may provide insights into the role of hyphae in pathogenesis.

The cell wall of Candida albicans is central to the yeasts ability to withstand osmotic challenge, to adhere to host cells, to interact with the innate immune system and ultimately to the virulence of the organism. Little is known about the effect of culture conditions on the cell wall structure and composition of C. albicans. We examined the effect of different media and culture temperatures on the molecular weight (Mw), polymer distribution and composition of cell wall mannan and mannoprotein complex. Strain SC5314 was inoculated from frozen stock onto yeast peptone dextrose (YPD), blood or 5 serum agar media at 30 or 37°C prior to mannan/mannoprotein extraction. Cultivation of the yeast in blood or serum at physiologic temperature resulted in an additive effect on Mw, however, cultivation media had the greatest impact on Mw. Mannan from a yeast grown on blood or serum at 30°C showed a 38.9 and 28.6 increase in Mw, when compared with mannan from YPD-grown yeast at 30°C. Mannan from the yeast pregrown on blood or serum at 37°C showed increased Mw (8.8 and 26.3) when compared with YPD mannan at 37°C. The changes in Mw over the entire polymer distribution were due to an increase in the amount of mannoprotein (23.8-100) and a decrease in cell wall mannan (5.7-17.3). We conclude that C. albicans alters the composition of its cell wall, and thus its phenotype, in response to cultivation in blood, serum and/or physiologic temperature by increasing the amount of the mannoprotein and decreasing the amount of the mannan in the cell wall.

No description available.

Occidiofungin is an antifungal compound that targets the conserved cytoskeletal protein, actin. Despite >90% amino acid conservation between fungal actin proteins, sensitivity to occidiofungin has been shown to vary with C. albicans, F. oxysporum, and P. digitatum exhibiting a resistant profile relative to S. cerevisiae. To determine whether differences in the amino acid sequences of actin contribute to differences in occidiofungin susceptibility, we expressed the actin gene from these fungal organisms in the ACT1 S. cerevisiae shuffle strain. Functionality of actin gene products was determined by measuring growth kinetics, actin protein levels, nuclear position, and actin cable formation. Results demonstrated functional complementation for all actin orthologs. Analysis of occidiofungin susceptibility found that fungal actin ortholog expression resulted in a similar sensitivity profile as the wildtype S. cerevisiae. These findings suggest that amino acid differences in actin are not directly responsible for the resistance to occidiofungin identified for these fungal organisms.

Mutações no gene CD40LG estão associadas à Síndrome de Hiper-IgM, relacionada ao cromossomo X, que é uma Imunodeficiência Primária. A ausência da interação CD40L/CD40 (linfócitos-DCs) acarreta um aumento da suscetibilidade às infecções fúngicas. Os efeitos do tratamento in vitro com interferon-gama em células dendríticas de pacientes com mutações no CD40L demonstraram potencial sobre a imunologia das DCs e nos remete a fundamentar possíveis ensaios clínicos futuros, que tenham por tema a prevenção e o tratamento de infecções oportunistas. / Mutations in the CD40LG gene are associated with the X-linked Hyper-IgM Syndrome, which is a Primary Immunodeficiency. The absence of the CD40L / CD40 interaction (lymphocytes-DCs) leads to increased susceptibility to fungal infections. The effects of in vitro gamma-interferon treatment on dendritic cells from patients with CD40L mutations have demonstrated potential for DC immunology and suggest that future clinical trials should address the prevention and treatment of opportunistic infections.

Made available in DSpace on 2015-05-14T12:56:05Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 1313077 bytes, checksum: 89fbd8d51a7f2a7e5318f325ec6d5c86 (MD5) Previous issue date: 2009-12-04 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The objective of this study was to make a correlation between the clinical, mycologic and histopathological diagnosis of buccal lesions, in carrying patients of dental prostheses, in order to verify the degree of agreement between the same ones. 47 individuals were examined, of both the sorts, taken care of in the Service of Health of the Centro Odontológico de Cruz das Armas (COCA), in João Pessoa - PB, the period of January the August of 2009. The data were collected by means of intraoral clinical examination, being written down in a chart treatment the type of prostheses, hygiene form, frequency and time of use of the prostheses, type of lesion and description of the same one. After surgical removal of the lesions, the pieces were led to the histopathological analysis, in the Buccal Pathology Laboratory of the UFPB, to confirm the clinical diagnosis. All the patients were submitted to the mycologic examination, being the harvested samples of the palatal mucous and the surface of the prostheses, with swabs. After, they were carried to the Mycology Laboratory of the UFPB. When compared the clinical diagnosis with the histopathological diagnosis, it can be observed that it had a correlation between the results, but in 03 (16,8%) cases of HFI and 02 (40%) cases of HPI the histopathological diagnosis was not compatible with the clinical diagnosis. In the clinical diagnosis of prostheses-induced stomatitis it had rightness of 90,6% (23 cases) in the mycologic examination, while the negative clinical diagnosis the 73,3% (11 cases) rightness. It was concluded that the presence of other unrelated injuries to the use of prostheses (Oral squamaus papilloma, pyogenic granuloma) reflects a bigger attention of the surgeon-dentist as for the distinguishing diagnosis of these buccal lesions, requesting, whenever possible, the histopathological and mycologic examinations, for definitive diagnosis. / O objetivo deste estudo foi fazer uma correlação entre diagnósticos clínico, histopatológico e micológico de lesões bucais, em pacientes portadores de próteses dentárias, a fim de verificar o grau de concordância entre os mesmos. Foram examinados 47 indivíduos, de ambos os gêneros, atendidos no Serviço de Saúde do Centro Odontológico Cruz das Armas (COCA), em João Pessoa PB, no período de janeiro a agosto de 2009. Os dados coletados, através de exame clínico intrabucal, foram anotados em uma ficha clínica, e consistiram do tipo de prótese usada, da forma de higiene, freqüência e tempo de uso da prótese, tipo de lesão e descrição da mesma. Após remoção cirúrgica das lesões, as peças foram encaminhadas para análise histopatológica, no Laboratório de Patologia Bucal da UFPB, para confirmação de diagnóstico clínico. Todos os pacientes foram submetidos ao exame micológico, sendo as amostras colhidas da mucosa palatina e da superfície da prótese, com swabs esterilizados. Em seguida, foram transportadas para o Laboratório de Micologia da UFPB. Quando comparado o diagnóstico clínico com o diagnóstico histopatológico, pode-se observar que houve uma correlação entre os resultados, porém, em 03 HFI (16,8%) e 02 HPI (40%) o diagnóstico histopatológico não foi compatível com o diagnóstico clínico. No diagnóstico clínico de estomatite protética houve acerto de 90,6% (23 caos) no exame micológico, enquanto o diagnóstico clínico negativo o acerto foi de 73,3% (11 casos). Concluiu-se que a presença de outras lesões não relacionadas ao uso da prótese dentária (papiloma escamoso oral, granuloma piogênico) reflete uma maior atenção do cirurgião-dentista no que se refere ao diagnóstico diferencial dessas lesões bucais, solicitando, sempre que possível, os exames histopatológico e micológico, para diagnóstico definitivo.

Candida albicans (C. albicans) constitui um fungo comum nas mucosas do trato gastrointestinal, incluindo cavidade bucal, que pode ocasionar candidose local ou invasiva, principalmente em estados de imunossupressão. Os mecanismos de defesa contra este fungo podem ser desencadeados pela ligação dos receptores de reconhecimento de padrões, TLR2 e dectina-1, aos seus ligantes, como a fosfolipomanana e os -glucanos encontrados na parede celular de C. albicans. Os mastócitos possuem estes receptores em sua membrana celular e residem nas interfaces com o ambiente, podendo constituir umas das primeiras linhas de defesa. Seus mecanismos imunes incluem síntese e secreção de mediadores, apresentação de antígenos, bem como atividades fagocitária e microbicida. Todos estes mecanismos de defesa podem ser desencadeados de forma independente ou cooperativa entre os receptores TLR2 e dectina-1. Deste modo, o objetivo deste trabalho foi avaliar in vitro a ocorrência de fagocitose, a geração de óxido nítrico e peróxido de hidrogênio pelos mastócitos desafiados ou não com C. albicans, e a participação do TLR2 e dectina-1 nesses eventos. Para isto, mastócitos, diferenciados da medula óssea (BMMCs) de camundongos selvagens (BMMCs Wt) ou TLR2-/- (BMMCs TLR2-/-) foram desafiados com C. albicans. Células eram também bloqueadas in vitro com anticorpos específicos anti-dectina-1(BMMCs BD-1 e BMMCs TLR2-/-/BD-1). Os eventos foram analisados por meio de ensaio fluorescente de fagocitose, método colorimétrico de Griess e pelos kits DAF-FM diacetato, Cell Rox Deep e Amplex Red. Os resultados foram expressos através de porcentagem, valores médios e desvios padrão, obtidos a partir de pelo menos três experimentos independentes. As análises estatísticas foram realizadas através do teste ANOVA fatorial, seguido de Fischer. Entre os BMMCs Wt, houve maior taxa de fagocitose com uma maior produção intracelular de NO aos 60 minutos em comparação aos outros tempos. A liberação extracelular de NO foi maior aos 120 minutos em relação aos outros tempos. O número de leveduras fagocitadas aumentou com o tempo, porém com diferença significante somente entre os tempos de 30 e 120 minutos. Entre os BMMCs TLR2-/-, houve maior número de leveduras fagocitadas aos 60 minutos em comparação aos 120 minutos. Porém, a liberação extracelular de NO foi menor aos 60 minutos em relação aos outros tempos. Comparando-se com os BMMCs Wt, os BMMCs TLR2-/- apresentaram uma redução na taxa de fagocitose, aos 60 minutos, menor liberação de NO extracelular, em todos os tempos, e menor número de leveduras fagocitadas aos 120 minutos. Comparando-se com os BMMCs Wt, os BMMCs BD-1 e os BMMCs TLR2-/-/BD-1 apresentaram uma redução na taxa de fagocitose com uma menor produção intracelular de NO, aos 60 minutos, e menor liberação de NO extracelular, aos 60 e 120 minutos. Comparando-se com os BMMCs Wt, os BMMCs TLR2-/-/BD-1 apresentaram uma maior produção de NO intracelular, aos 30 minutos, e menor número de leveduras fagocitadas aos 60 e 120 minutos. Sendo assim, concluímos que os mastócitos são capazes de fagocitar C. albicans com concomitante produção de substâncias potencialmente candidacidas. Concluímos também que estes mecanismos envolvem o reconhecimento do fungo via TLR2 e dectina-1, principalmente de forma sinérgica. / Candida albicans (C. albicans) is a common fungus present in gastrointestinal tract mucosa including oral cavity, which may cause local or invasive candidiasis, especially in immunosuppression. The mechanisms of defense against this fungus may be triggered by the binding of the pattern recognition receptors TLR2 and dectin-1 to its ligands, such as phospholipomannan and -glucans found in the cell wall of C. albicans. Mast cells express these receptors on cell membrane and reside in the interfaces with the environment, and may be one of the first lines of defense. Their immune mechanisms include synthesis and secretion of mediators, antigen presentation, as well as phagocytic and microbicidal activities. These mechanisms can be triggered independently or cooperatively by TLR2 and dectin-1. Therefore, the aim of the study was to evaluate in vitro the phagocytosis, the generation of nitric oxide and hydrogen peroxide by mast cells challenged or not with C. albicans, and the involvement of TLR2 and dectin-1 receptors in these mechanisms. Bone marrow-derived mast cell (BMMCs) from wild type mice (BMMCs Wt) or TLR2-/- (BMMCs TLR2-/-) was challenge with C. albicans. Cells were also in vitro blocked with specific anti-dectin-1 antibodies (BMMCs BD-1 and BMMCs TLR2-/-BD-1). The mechanisms were analyzed using fluorescent phagocytosis assay, Griess colorimetric method and by DAF-FM diacetate, CellRox® Deep Reagent and Amplex® Red enzyme assays. Results were expressed by percentage, mean and standard deviations obtained from the least three independent experiments. Statistic was performed using factorial ANOVA and Fischer. Among BMMCs Wt, there was higher phagocytosis rate associated with increased intracellular NO production at 60 minutes, comparing to other periods. The extracellular release of NO was higher at 120 minutes comparing to other periods. The number of phagocytized yeasts increased over time, however with significant difference only among the 30 and 120 minutes. Among BMMCs TLR2-/-, there was higher number of phagocytized yeast at 60 minutes compared to 120 minutes. However, the extracellular release of NO at 60 minutes was lower comparing to other periods. In comparison to BMMCs Wt, the BMMCs TLR2-/- showed a reduction in the phagocytosis rate, at 60 minutes, lower release of extracellular NO, at all times, and fewer numbers of phagocytized yeast at 120 minutes. Compared to BMMCs Wt, the BMMCs BD-1 and BMMCs TLR2-/-/BD-1 showed a reduction in the phagocytosis rate with lower intracellular NO production, at 60 minutes, and decrease of extracellular NO release, at 60 and 120 minutes. Comparing to BMMCs Wt, the BMMCs TLR2-/-/BD-1 showed increased production of intracellular NO, after 30 minutes, and fewer phagocytized yeast, at 60 and 120 minutes. Therefore, we conclude that mast cells are able to phagocytose C. albicans with concomitant production of the potentially microbicidal substances. Also conclude that these mechanisms involve the fungal recognition via TLR2 and dectin-1, especially by means of synergistic way.