The main aim of this project was to determine the potential of increased chitin content as a mechanism of resistance to caspofungin in different fungal pathogens. <i>C. albicans</i> wild-type cells were pre-grown with a combination of CaCl₂ and CFW prior to caspofungin treatment. This result sin a three-fold increase in cell wall chitin. Wild-type cells, which had elevated chitin content, were less susceptible to caspofungin. Priming cells to activated chitin synthesis was also able to compensate for the loss of the normally essential <i>CaCHS1</i>, through formation of three novel forms of salvage septa. In the absence of both <i>Ca</i>Chs1 and <i>Ca</i>Chs3, which are typically involved in septum formation, the class I chitin synthases, <i>Ca</i>Chs2 and <i>Ca</i>Chs8, could be stimulated to synthesise a proximally offset salvage septum. When <i>Ca</i>Chs3 was the only remaining chitin synthase, treatment with CaCl₂ and CFW, led to the formation of thick chitin-rich salvage septa. <i>Ca</i>Chs2<i> </i>and <i>Ca</i>Chs3 could be stimulated by treatment with CaCl₂ and CFW to synthesise a thin salvage septum similar to the septum of wild-type cells. All three salvage septa were capable of restoring viability and cell division in <i>C. albicans.</i> The compensatory increase in chitin content in response to caspofungin treatment was not specific to <i>C. albicans</i> because clinical isolates of <i>C. tropicalis, C. parapsilosis </i>and <i>C. guilliermondii</i> and the filamentous fungus, <i>A. fumigatus</i>, also demonstrated an increase in chitin content after treatment with caspofungin. Isolates of <i>C. glabrata</i> and <i>C. krusei</i> showed no change in chitin content when exposed to caspofungin. The results of this thesis highlight the potential for using chitin synthase inhibitors in combination therapy with the echinocandins.

Organisms exist in complex and dynamic environments. Facing numerous challenges, microorganisms must continuously monitor environmental changes and adapt to these if they are to survive. For example, the major fungal pathogen of humans, Candida albicans experiences diverse stresses, including temperature fluctuations, oxidative stress and enzymic processes, that cause molecular damage. Post-translational modifications such as phosphorylation play major roles in stress adaptation, for example through activation of MAP kinase pathways and transcription factors such as the heat shock transcription factor, Hsf1. However, other post-translational modifications such as ubiquitination and sumoylation have been relatively understudied. Nevertheless, they are believed to play crucial regulatory roles in many cellular processes including stress adaptation. Therefore, in this study the roles of ubiquitin and SUMO (small ubiquitin-like modifier) have been investigated in C. albicans. Proteomics was used to identify ubiquitination and sumoylation targets, and this was combined with molecular analyses of the UBI4 and SMT3 genes, which encode polyubiquitin and SUMO, respectively. Both ubiquitination and sumoylation were shown to play important roles in morphogenesis, cell division and stress adaptation in C. albicans, including adaptation to heat and oxidative stresses. In addition, the dynamics of heat shock adaptation were examined in C. albicans using a systems biology approach. Hsf1 is known to activate HSP90. In this study, Hsf1 was found to be transiently phosphorylated in response to heat shock, and Hsp90 was found to down-regulate this Hsf1 phosphorylation. This led to the identification of an autoregulatory loop that controls thermal adaptation in C. albicans. A mathematical model of heat shock regulation (constructed in collaboration with Katarzyna Tyc and Edda Klipp) provided novel insights into the regulation of this evolutionarily conserved environmental response and the significance of thermal adaptation during systemic Candida infection.

Candida albicans is the major fungal pathogen of humans and its cell surface mannoproteins play important roles in adhesion, interactions with the host and signal transduction. Most of the covalently attached mannoproteins are linked to the cell surface by glycosylphosphatidylinositol (GPI) anchors. Over 100 putative GPI-proteins have been identified in silico in the C. albicans genome, yet the majority remain uncharacterised with regards to their function and regulation. This study uses different approaches to define the role and regulation of selected GPI-protein genes. Expression analyses demonstrated transcriptional regulation of these genes in response to membrane and wall stresses including antifungal drugs. We propose that these genes encode proteins with important roles in cell wall remodelling. One protein, Pga54 was selected for further analysis. Null mutant and reintegrant strains were constructed and phenotypically analysed. The mutant phenotype was for the greatest parts inconclusive, but it showed a minor virulence defect in a mouse model of systemic infection. Using a tagged version of Pga54 the protein could be detected in the GPI fraction of the cell wall in wild type C. albicans cells and was shown to be higher expressed in cells subjected to stress. To establish whether they play a role in adhesion to host cells selected uncharacterised GPI proteins were heterologously expressed in the non-adherent yeast Saccharomyces cerevisiae. The newly generated S. cerevisiae strains did not show any differences in adhesion to human buccal epithelial cells. Further, the examination of tandem repeat variation in genes encoding GPI anchored proteins in different C. albicans strains indicates that this is could be a further means of creating diversity and heterogeneity at the cell surface. This study employs different methods to study the role and the dynamics of cell surface proteins. In future studies these approaches could be applied to all identified GPI proteins to obtain a comprehensive picture of cell surface diversity in Candida albicans.

Mini-dissertation submitted in partial compliance with the requirements of the Master's Degree in Technology: Homoeopathy, Durban Institute of Technology, 2002. / The purpose of this study was to determine the efficacy of certain Compositae species herbal extracts (Arctium lappa, Calendula officinalis and Echinacea purpurea) in the inhibition of in vitro growth of Candida albicans as compared to nystatin in terms of the disc diffusion test. Candida albicans was obtained from the Department of Biotechnology (Technikon Natal). Three components were tested on Candida albicans: namely the herbal extracts which were the experimental group, nystatin which was the allopathic component serving as a positive control and the 62% ethanol which was the negative control. Commercially available herbal extracts of Arctium lappa, Calendula officinalis and Echinacea purpurea respectively, with an ethanol concentration of 62% v/v, were purchased from Parceval (Pty) Ltd. The herbal extracts were prepared according to the German Herbal Pharmacopoeia (1991) standards. Commercially available nystatin suspension was obtained from \ Bristol-Myers Squibb (Pty) Ltd, 62% (vlv) ethanol was prepared according to the German Homoeopathic Pharmacopoeia (1991) standards. In carrying out the experiment, 5mm filter paper discs were placed on Sabouraud's dextrose agar plates which were streaked with Candida albicans. Thereafter 0.7 microlitres of the test and control substances were pipetted onto / M

The aim of the project was to determine the sequence of the gene encoding glucose-6-phosphate dehydrogenase (G6PDH) in the industrially used, but genetically little explored yeast Candida utilis. In the first strategy, a C. utilis DNA library was constructed in the expression vector phage lambda gt11, and was screened immunologically with antiserum raised against C. utilis G6PDH. The second strategy involved construction of libraries in the plasmid vector pTZ18R and screening with a mixed oligonucleotide probe. Two methods were developed that allow DNA, of appropriate quality for the construction of DNA libraries, to be prepared from C. utilis cells. Both involve centrifugation of C. utilis lysates through caesium trifluoroacetate gradients. A rabbit antiserum was raised against a commercial preparation of C. utilis G6PDH. It reacted with Western blotted C. utilis G6PDH, and non-specific binding of the antiserum to Western blotted E. coli protein was reduced by treatment with an E. coli extract. Antiserum pretreated in this way was used for immunological screening. Sequence information was determined for the inserts of 7 phage identified during immunological screening. None of the insert sequences was considered to contain part of the C. utilis G6PDH coding sequence. One putative positive colony was identified during the screening of the pTZ18R libraries. Sequence determination showed the insert to contain a region complementary to 16 bases in one of the 17-base long probe oligonucleotides. This insert was considered not to contain part of the C. utilis G6PDH coding sequence. Time did not permit further work which, using the methods developed, should now permit determination of the C. utilis G6PDH gene sequence.

Trabajo de Investigación Requisito para optar al Título de Cirujano Dentista / Autor no autoriza el acceso a texto completo de su documento / Las levaduras del género Candida son patógenos oportunistas que se encuentran como comensales en la cavidad oral. Candida glabrata ha emergido como un importante agente patogénico en la mucosa oral. Recientemente, se ha descrito la colonización por levaduras en pacientes con periodontitis, estimándose que aproximadamente un 20 % de éstos portan levaduras en sacos periodontales. Hay pocos estudios que identifiquen aislados de C. glabrata en sujetos con periodontitis. En un estudio previo realizado en el laboratorio de Bioquímica y Biología Oral, se identificaron presuntivamente 60 aislados de la especie C. glabrata en diversos nichos orales de sujetos periodontalmente sanos y pacientes con periodontitis crónica y agresiva. El propósito de este estudio fue corroborar la identidad de las especies presuntivamente identificadas y medir la variabilidad genética y diversidad genotípica de estos aislados dentro y entre individuos. La re-identificación de levaduras se realizó mediante la prueba bioquímica API ID32C y la técnica de PCR-RFLP, amplificando la región ITS1-5.8S-ITS2 rDNA y cortando el amplicón con la enzima MspI. La variabilidad genética, la diversidad genotípica y el análisis de agrupamiento se realizaron mediante la técnica de RAPD-PCR, usando dos partidores de la serie operón y la asistencia del programa computacional TreeCon. De los 60 aislados analizados, sólo el 93,3 % fue re-identificado como C. glabrata, el resto, 6,7 %, correspondió a 4 aislados de un sujeto periodontalmente sano que fueron identificados como C. albicans. El coeficiente de similitud promedio ( S ) fue de 0,849. Se obtuvieron 10 genotipos en total y dos clusters genotípicos principales, constituidos por aislados no relacionados entre sí, evidenciando heterogeneidad genética de éstos dentro y entre individuos. El mayor S promedio y la mayor diversidad genotípica ( G ) fueron encontrados en los aislados de levaduras recuperados de un paciente con periodontitis crónica y agresiva, respectivamente. La realización de este estudio es importante porque permitirá aportar al conocimiento de esta especie, sus características fenotípicas y genéticas y ayudar AB 1 AB así a esclarecer la posible relación entre la presencia de levaduras del género Candida en sitios subgingivales enfermos.

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%.

During the infection process Candida albicans has to respond to various stresses imposed by host environment including oxidative and osmolarity stress generated by phagocytic cells such as macrophages and neutrophils. Exposure to caspofungin and other antifungal antibiotics also imposes stress on the C. albicans cell wall. These various stress responses are orchestrated through the activation of multiple stress pathways including the cAMP-PKA, several MAPK pathways and the Ca2+-calcineurin pathway which influence cell wall shape and composition. Such changes were predicted to influence recognition of C. albicans by innate immune cells. During my Ph.D. studies I primarily investigated the effect of the activation or inhibition of these pathways on the interaction with the innate immune cells by examining phagocytosis, the cytokine profile induced by mononuclear and polynuclear cells of the innate immune system. I found that the activation and inhibition of these pathways plays an important role in remodeling of cell wall and hence the immunological profile. Inactivation of cAMP, Calcium signaling pathway by the deletion of TPK1 and CNA1 resulted in marked reduction in pro-inflammatory cytokine production. Inactivation of MAPK pathway by deletion of HOG1 altered major pro-inflammatory cytokine secretion. Cytokine production was also affected by exposure of C. albicans signaling mutants to Calcofluor White, caspofungin, oxidative and osmotic inducing stresses. Cytokine stimulation was also affected by deletion of URA3, exposure of C. albicans to rifmapicin and antimycin A. These results suggest that stress signaling pathways act to regulate collateral changes in the cell wall, which in turn affects the immune reactivity. Pro and anti-inflammatory cytokine and antifungal profiles of Candida haemulonii was also found to be highly variable. Thus regulation and exposure to different microenvironments significantly modifies immunological signature of fungal cells, suggesting that responses to local stresses make the fungal cell surface a moving target for immunological surveillance.

Trabajo de Investigación Requisito para optar al Título de Cirujano Dentista / En Chile el 63,2% de la población mayor de 65 años usa prótesis removible. Cuando el estado protético no cumple con requisitos funcionales, produce lesiones en la mucosa oral y facilita la formación de biofilm en la superficie protésica. El uso de prótesis removibles no funcionales conduce al desarrollo de estomatitis protésica, un proceso inflamatorio de la mucosa de soporte de diversa extensión y severidad, que tiene como factor etiológico principal la presencia e infección por Candidas. El objetivo de esta investigación fue determinar presencia, cantidad y especies de Candida spp y su asociación con la estomatitis protésica en portadores de prótesis removible antes (con prótesis no funcionales) y después del tratamiento rehabilitador (con prótesis nuevas y funcionales). Material y métodos Se realizó un estudio descriptivo cuantitativo, con 34 pacientes, 69 años como edad promedio, portadores de prótesis removible no funcionales, con y sin estomatitis protésica atendidos en la Clínica Odontológica Universidad de Chile. Se tomó muestras de saliva antes y después del tratamiento rehabilitador, sembrándolas en agar Sabouraud para verificar presencia, realizar recuento e identificación de levaduras del género Candida. Las variables fueron analizadas con el test de correlación de Pearson con corrección de Yates, test exacto de Fischer o test Kruskal Wallis según correspondiese. 5 Resultados La estomatitis protésica se presentó en 55,9% de los pacientes, correspondiendo 29,4% a tipo I, 26,5% tipo II, y sin presencia de tipo III en este estudio. La presencia y recuento de Candidas fueron mayores en aquellos con estomatitis protésica tanto antes como después del tratamiento rehabilitador. Al tener prótesis nuevas y funcionales el recuento disminuyó de manera significativa en todos los pacientes, sin embargo permaneció alto en aquellos con estomatitis protésica. La especie identificada con más frecuencia fue Candida albicans. Conclusiones Se evidenció una alta presencia de estomatitis protésica en pacientes portadores de prótesis removible no funcionales, siendo un alto recuento de Candidas un factor de riesgo para el desarrollo de esta lesión. Al cambiar las prótesis removible no funcionales por nuevas y funcionales, el conteo salival de Candidas disminuye, demostrando asociación entre recuento de levaduras del género Candida y desarrollo de estomatitis protésica cuando el estado protésico no cumple con los requisitos funcionales. Palabras claves: Prótesis removible, estomatitis protésica, levaduras del género Candida.

Trabajo de Investigación Requisito para optar al Título de Cirujano Dentista / Autor no autoriza el acceso a texto completo de su documento / Introducción El vertiginoso aumento de la población adulta mayor en Chile, hace relevante el estudio de las patologías más comunes en este grupo. La estomatitits protésica (EP) es una de las lesiones orales más prevalentes en sujetos portadores de prótesis removible (PR) y se define como un proceso inflamatorio crónico de la mucosa adyacente al aparato protésico. La etiología de la EP es multifactorial, pero se asocia en un alto porcentaje a levaduras del género Candida, denominándose en estos casos como candidiasis oral. Otros de los factores que han sido relacionados con la enfermedad, son alteraciones en parámetros salivales como velocidad de flujo salival (VFS), pH y concentración total de proteínas en la saliva, además de la presencia de xerostomía. La evidencia sostiene que éstos podrían jugar un rol en la patogenia de la EP asociada a candidiasis. El propósito de este estudio fue caracterizar dichos parámetros salivales en sujetos adultos mayores portadores de PR con EP asociada a candidiasis y compararlos con un grupo de sujetos portadores de PR sin la enfermedad. Metodología El presente estudio incluyó 70 voluntarios mayores de 60 años pertenecientes a un establecimiento de larga estadía para adultos mayores (ELEAM) y a la clínica de prótesis totales, de la Facultad de Odontología de la Universidad de Chile. La muestra se dividió en 2 grupos, el experimental, constituído por pacientes con EP asociada a candidiasis, y el control, quienes no presentaban dicha enfermedad, ambos portadores de PR. Previa firma del consentimiento informado, la recolección de datos se realizó mediante una ficha clínica en donde se registró la presencia de xerostomía, y se obtuvieron muestras salivales, las cuales fueron analizadas para determinar VFS, pH y concentración total de proteínas en la saliva. Resultados No hubo diferencias estadísticas entre la VFS, prevalencia de xerostomía y concentración total de proteínas en la saliva entre ambos grupos de estudio. En cuanto al pH, éste fue menor en el grupo experimental comparado con el grupo control, con diferencia estadística. Además la VFS fue menor en el grupo con xerostomía que en el grupo que no presentó el síntoma. 2 Conclusiones Existe poca evidencia respecto a los parámetros salivales y su relación con la EP asociada a candidiasis. En este estudio solo se encontró diferencia significativa en el pH de los pacientes con y sin EP, sin embargo, son necesarios más estudios que aíslen las variables confundentes para poder dilucidar el rol de la saliva en la patogenia de la enfermedad, y así, conocer sus implicancias en el diagnóstico y terapéutica de la EP asociada a candidiasis.