No description available.

El propósito del presente fue de identificar las especies de Candida implicadas en estomatitis subprotésica en pacientes del Departamento de Odontoestomatología del Centro Médico Naval “CMST” – 2007 Se analizaron los 30 primeros pacientes con diagnóstico de estomatitis subprotésica que acudían al Departamento, a los cuales se les realizó cuatro frotises, dos para el examen directo microscópico (con coloración Gram) para confirmar la presencia de levaduras, y dos para el cultivo en Agar Sabouraud+Cloranfenicol, del crecimiento en este agar, se hizo la prueba del tubo germinal para determinar la presencia de Candida albicans, de salir negativo esta prueba, se llevaba a cabo la identificación de la especie mediante el sistema Api Candida. Se obtuvieron entre otros resultados que Candida albicans fue la especie más implicada en la estomatitis subprotésica con un 96.66% seguido de Candida tropicalis con un 3.33%. / The purpose of this was to identify the species of Candida involved in stomatitis subprotésica in patients of the Department of Dental Naval Medical Center "CMST" - 2007 We analyzed the first 30 patients diagnosed with stomatitis subprotésica who went to the Department, to which were conducted four frotises, two for the direct microscopic examination (colouring Gram) to confirm the presence of yeast, and two to be planted in Agar Sabouraud + Chloramphenicol, growth in the agar, it was the germ tube test to determine the presence of Candida albicans, to leave this negative test was carried out to identify the species by the API system Candida. Among other results were obtained that Candida albicans was the most involved in stomatitis subprotésica with a 96.66% followed by Candida tropicalis with a 3.33%.

Thesis supervisor: David R. Soll. Includes bibliographic references (p. 280-316).

published_or_final_version / Medicine / Master / Master of Medical Sciences

Chitin biosynthesis in the fungal cell wall is essential for cell viability. Chitin play roles in maintaining cellular integrity and up regulation of chitin synthesis helps protect the cell from cell wall stresses or cell wall damaging antifungal agents such as caspofungin. In Candida albicans chitin is synthesised by four chitin synthases (Chs), Chs1, Chs2, Chs3, and Chs8. The biological function of Chs2 and Chs8 (class I chitin synthases) is less well understood. The major objective of this thesis was to understand the biological function of the class I chitin synthases. Chs2-YFP and Chs8-YFP showed a dynamic localisation at septation sites, which were first visualised as a bar which then contracted to a spot. This spot then separated into two spots, one on each sides of the septum. These two spots remained there until yeast cell separation, and remained at this location throughout several subsequent hyphal cell cycles. Chs2-YFP also localised to hyphal tips. The phenotype of a chs2Δchs8Δ double mutant was re-investigated using the propidium iodide. Intact dead germ tubes and hyphal tip lysis was observed in a chs2Δchs8Δ mutant cells. This suggested that Chs2 and Chs8 play a major role in the maintenance of hyphal tip integrity and polarised growth and perhaps a minor role in septum formation. Studies were also performed to assess whether phosphorylation regulated the localisation of Chs2-YFP. It was shown that the localisation of Chs2-YFP to septation sites was regulated by phosphorylation on S222. A version of Chs2-YFP that could not be phosphorylated (Chs2S222A-YFP) localised at the septa in lower amounts than the Chs2-YFP, and a version of Chs2-YFP that mimicked constitutive phosphorylation (Chs2S222E-YFP) localised normally. This suggested that phosphorylation of Chs2 on S222 facilitates the localisation of Chs2-YFP at septation sites, and that dephosphorylation is not required for this cellular localisation. In the presence of cell wall stresses (CaCl2/CFW) and caspofungin, more Chs2-YFP was observed and the average intensity of fluorescence of Chs2-YFP was higher in the presence of these stresses than in untreated cells.

Fungal nitrosative stress responses are understudied and poorly understood. Also, while the responses of the pathogen Candida albicans to individual oxidative and cationic stresses have been characterised relatively well, the responses of this pathogen to simultaneous combinations of these and nitrosative stresses have not been elaborated. Yet, C. albicans is exposed to these combinatorial stresses during disease progression. Therefore, the overall aim of this project was to characterise the "Nitrosative and Combinatorial Stress Responses in Candida albicans". The first objective was to elucidate nitrosative stress response mechanisms in C. albicans at a cellular, transcriptional and post-translational level. Genetic screens revealed that the Hog1 and Mkc1 signalling pathways contribute to nitrosative stress adaptation in addition to Cta4 signalling. The next objective was to characterise responses to combinatorial nitrosative, oxidative and/or cationic stresses with respect to the relevant signal transduction pathways. Unexpectedly, combinatorial stresses did not activate classical nitrosative (Cta4) and oxidative (Cap1) signalling mechanisms. Instead, Hog1 signalling was found to play a significant role in the combinatorial stress adaptation. Dramatic changes in the GSH redox potential were observed in response to both single and combinatorial stresses. Thus, GSH redox regulatory mechanisms were explored in more detail. BLAST searches of the C. albicans genome were performed, revealing genes encoding a putative NADPH-dependent glutathione reductase (Glr1) and a GSH-dependent S-nitrosoglutathione reductase (Fdh3). We confirmed that both conserved enzymes are involved in the maintenance of redox homeostasis and hence are crucial for C. albicans adaptation during host-pathogen interactions. This was achieved by showing that inactivation of Glr1 or Fdh3 disturbs GSH redox homeostasis, confers oxidative and formaldehyde stress sensitivity and attenuates virulence.

Candida albicans is the most common systemic fungal pathogen of humans. Virulence factors, fitness attributes and the host immunity ultimately determine the ability of C. albicans to reside as a commensal organism or an opportunistic pathogen in its human host. C. albicans displays a high level of flexibility in adapting to and infecting diverse niches in its human host. Generally occupying glucose-poor niches, C. albicans often depends upon alternative carbon sources to grow and establish infections. However, most cellular and molecular investigations of this pathogen have been performed on glucose-grown cells, and the relationship between physiological nutrients and drug resistance or virulence has not been studied. The main objective of this thesis was to study the effects of host carbon sources upon C. albicans cell biology and how this in turn affects the stress and drug resistance, immune recognition and virulence of this fungus. Growth on this physiologically relevant alternative carbon source was shown to influence the cell wall architecture of C. albicans and alter its resistance to a variety of stresses and antifungal drugs. Differences in stress resistance were less dependent on the key stress signalling pathways than on major architectural changes in the C. albicans cell wall that influenced its biochemical and biophysical properties. It was also shown that carbon source has a major impact upon C. albicans pathogenicity, altering cell adhesion and host virulence, in both systemic and vaginal infections. Considering the different niches that Candida can colonise in the body, these findings have the potential to change our views about the interactions that occur between the fungus and its human host. Taken together, the results of this project demonstrate that differential nutrient availability within diverse host niches influences the ability of pathogenic Candida species to counteract local chemical insults and pharmacological interventions.

No description available.

published_or_final_version / Dentistry / Doctoral / Doctor of Philosophy

It has been envisaged that lytic enzymes may be crucial in determining the morphology and growth of fungi, and may therefore represent a target for antifungal agents. The chitinolytic system of <i>Candida albicans</i> was investigated using a range of 4-methylumbelliferyl glycosides as model substrates, and its potential as a target for antibiotics has been assessed. Maximum hydrolysis was obtained with substrates having monomer and tetramer chain lengths, being attributed to <i>N</i>-acetylglucosaminidase and endochitinase respectively. Activities were investigated in cell fractions, vacuoles, and also in whole cell preparations. The characteristics of both chitinase and <i>N</i>-acetylglucosaminidase were examined, including pH and temperature optima and Km values. Chitinase was semi-purified on Fast Protein Liquid chromatography system and activity could be located after native polyacrylamide gel electrophoresis. Analysis of chitinase activity during the growth of the yeast morphology of <i>C.albicans</i> revealed maximal activities during the logarithmic phase, suggesting a relationship of chitinase levels to active growth of the pathogen. It was found that the antibiotic allosamidin was a potent inhibitor of chitinase activity of <i>C.albicans</i>, but not of <i>N</i>-acetylglucosaminidase. Conversely, an analogue of <i>N</i>-acetylglucosamine was found to be a potent inhibitor of <i>N</i>-acetylglucosaminidase but not of chitinase. Treatment of yeast suspensions with allosamidin resulted in an increased chain length. No cell death, or discernible pattern of change in the radiolabelling was observed in the presence of either allosamidin or <i>N</i>-acetylglucosamine analogue. Similarly no consistent change in the optical density of cultures was observed in the presence of either inhibitor. Even in the presence of the membrane permeabilising agent amphotericin no effects were observed above those achieved with amphotericin alone. Comparative studies were carried out upon the chitinolytic activity of <i>Kluyveromyces lactis</i> toxin and bovine serum. The chitin synthetic system of <i>Benjaminiella poitrasii</i> was compared to that of <i>C.albicans</i>.