Thesis (M.Tech.: Homoeopathy) - Dept. of Homoeopathy, Durban Institute of Technology, 2004 xxvii, 155 leaves / The aim of this in vitro study was to determine the effect of Commiphora molmol tincture prepared in 86% v/v ethanol, Hydrastis canadensis tincture prepared in 62% v/v ethanol and Warburgia salutaris tincture prepared in 62% v/v ethanol against Candida albicans (C. albicans) with 62% v/v ethanol, 86% v/v ethanol and fluconazole as the control agents, and to determine the Minimum Inhibitory Concentration/s (MIC/s) of the effective tincture/s using the disc diffusion assay.

The critical prompt innate immune response is highly built upon the influx of neutrophils from the blood stream to the site of infection. In the battlefield, neutrophils sense pathogen-associated molecular patterns (PAMPs) through their pattern-recognition receptors (PRRs) to launch a number of responses with the goal to defeat the invading pathogen. Neutrophils’ wide spectrum of responses ranges from reactive oxygen species production (ROS), phagocytosis, cytokine and chemokine secretion, and neutrophil extracellular trap (NET) formation. The NET scaffold is composed of nuclear chromatin which is armed with antimicrobial proteins. DNA traps are able to ensnare and kill microbes in the extracellular space and NET release concurs with cell death of the neutrophil. An increasing body of literature describes that NETs impose deleterious effects on the host itself in addition to their antimicrobial activity. These hazardous effects mainly stem from pro-inflammatory and tissue-destructive activity of NETs. These two diverse outcomes of NETs result in a series of effects on both host and pathogen. Therefore, it seems rational that NET formation is tightly regulated and not happening spontaneously. The opportunistic fungal pathogen Candida albicans captured and killed by NETs. This fungus has the remarkable ability to grow as budding yeast or as filamentous hyphae, and reversibly alternate between these morphotypes. Hyphae are the tissue-destructive, invasive and pro-inflammatory form of C. albicans, whereas yeast is the proliferative, non-invasive form. Hence, it is important to find out how neutrophils discriminate between distinct growth forms of C. albicans and how NET release is regulated in this regard. To assess neutrophils responses towards each growth form of C. albicans, the mere ratio of each fungal morphotypes is an insufficient measure to describe comparable amounts used in infection experiments; we therefore used dry mass of fungal cells to serve as a common denominator for amounts of fungal cells with different morphotypes. As assessment of dry mass is laborious, we developed a quick correlative method, which quantified fungal metabolic activity corresponding to the actual dry mass. We applied this method in consecutive studies investigating the neutrophil responses specific to different morphotypes of C. albicans. Positive and negative regulators of NET formation were investigated for this thesis in a mechanistic fashion. To identify how NET release is negatively regulated during C. albicans infection we focused on anti-inflammatory receptors on neutrophils. We observed that adenosine signals via adenosine receptor reduces the amount of NETs exclusively in response to C. albicans hyphae, the invasive, pro-inflammatory form. We identified adenosine receptor A3 as the responsible receptor suggesting that targeting of adenosine A3 would be a promising approach to control invasive fungal infection, since particularly during immune reconstitution invasive mycoses are frequently accompanied by hyperinflammation which additionally worsens the patient’s state. As unbalanced inflammation is harmful to the host, a situation reflected in autoimmune diseases, such as systemic lupus erythematosus, we aimed to find molecules, which are able to inhibit NET formation. Thus, we introduced the non-toxic agent tempol’’. During ROS-depended stimulation of NET formation via C. albicans and phorbol esters, the stable redox-cycling nitroxide tempol efficiently blocked NET induction. We therefore proposed tempol as a potential treatment during inflammatory disorders where NET formation is out of balance. In quest for positive regulators of NET formation we found the major addictive component of tobacco and electronic cigarettes, nicotine, as compelling direct inducer of NET release. Interestingly, nicotine is associated with exacerbated inflammatory diseases exerting its pro-inflammatory activity via acetylcholine receptor by targeting protein kinase B (known as Akt) activation with no effect on NADPH oxidase complex in a ROS independent fashion. In consideration of neutrophils role in smoking-related diseases we propose targeting Akt could lower the undesirable effect of NET.  In conclusion, this thesis identified new modulators of NET formation in response to fungal infection and more broadly to other NET-inducing stimuli, which might have implications in forthcoming therapies.

published_or_final_version / Oral Biology / Doctoral / Doctor of Philosophy

The human pathogen Candida albicans is polymorphic, and its ability to switch growth forms is thought to play an important role in virulence. The primary research aim of this thesis was to understand the role the mitotic exit network plays in C. albicans with particular focus on the Tem1 GTPase protein. This aim was split into three specific goals; to study the role of Tem1 through the construction of a regulatable tem1 mutant, to understand the regulation of Tem1 through localisation and protein interaction studies, and to construct new molecular tools utilising the NAT1 positive selection marker in order to achieve two previous goals. In this thesis we demonstrated that TEM1 is an essential gene in C. albicans, and its essential function is signalled through the Cdc15 protein. Surprisingly, Tem1p depleted cells arrested as hyper-polarised filaments containing one or two nuclei and ultimately lost viability. These filaments formed from budding yeast cells, suggestive of a blockage late in the cell cycle. Ultimately the failure of these filaments to undergo cytokinesis was linked to a defect in septin ring dynamics and the formation of actomyosin ring. To understand the regulation of Tem1 we localised both the Tem1 and Lte1 proteins and found that Tem1 localised to spindle pole bodies in a cell-cycle dependent fashion by recruited at the onset of S phase. In contrast, the Lte1 homolog localised to the daughter cell cortex prior to release into the cytoplasm at the end of the cell cycle. A yeast 2-hybrid analysis of the MEN components demonstrated the potential of Bfa1/Bub2 and Tem1 to form a complex and the ability of Tem1 to homodimerise which may play a role in its self-activation. In order to carry out various aspects of this work we constructed a fully functional set of cassettes, including the constitutively active ENO1 promoter, V5-6xHIS epitope tag and various fluorescent protein genes fused to the NAT1 positive selection marker. When considered together, these results indicate that Tem1 is required for timely mitotic exit and cytokinesis in C. albicans, similar to S. cerevisiae, but the final output of the pathway must have diverged.

Trabajo de Investigación Requisito para optar al Título de Cirujano Dentista / Autor no autoriza el acceso a texto completo de su documento / Introducción: La estomatitis protésica (EP) es una patología frecuente que afecta a sujetos portadores de prótesis removible (PR). Ésta se caracteriza por la inflamación y eritema de la mucosa adyacente a la PR y su etiología ha sido relacionada a múltiples factores. El principal de ellos es la presencia de recuentos elevados de levaduras del género Candida, sin embargo, el aumento de este microorganismo sería determinado por otras variables, las cuales dependen del hospedero, de la misma levadura, así como del microambiente oral. Esto a su vez, determina mayor complejidad en su tratamiento, el cual ha sido objeto de múltiples investigaciones en los últimos años. El objetivo de esta investigación es determinar las características microbiológicas y salivales de adultos mayores portadores de prótesis removible con y sin estomatitis protésica, al inicio del proyecto al cual este estudio está adscrito, denominado “Efecto del consumo de bebidas lácteas enriquecidas con probióticos en la reducción de incidencia de candidiasis oral asociada a estomatitis protésica, en adultos mayores portadores de prótesis removibles”. Materiales y métodos: Este estudio se realizó con 63 adultos mayores portadores de PR, pertenecientes a una institución de cuidados continuos y a la clínica de Prótesis Totales de la Facultad de Odontología, Universidad de Chile. Previa firma de consentimiento informado, a cada sujeto se le realizó una ficha clínica diseñada y validada para el estudio, tras lo cual fueron tomadas muestras de saliva no estimulada para su procesamiento y análisis. Las variables en estudio corresponden a Xerostomía, velocidad de flujo salival (VFS), pH, portación, recuento e identificación de Candida y recuento de Lactobacillus. Los datos se analizaron con los test X 2 , correlación lineal de Pearson, Test de Wilcoxon y Ttest, considerando un valor de p< 0,05. Resultados: De las características salivales analizadas, solo el pH exhibió diferencias estadísticas entre ambos grupos (p=0,0), no así Xerostomía y VFS. Adultos mayores con EP presentaron mayor portación y recuento de levaduras del género Candida (p=0,0), siendo C. albicans la especie más frecuentemente aislada en ambos grupos de estudio. En cuanto a los recuentos de Lactobacillus, estos fueron mayores en los sujetos sin EP (p=0,02). No hubo asociación entre los distintos parámetros salivales y recuentos de Candida o Lactobacillus. Conclusiones: Las características salivales carecen de un rol importante en la variación de los recuentos salivales de Candida y Lactobacillus en adultos mayores portadores de PR con y sin EP. Los recuentos elevados de C. albicans, representan un factor de riesgo para el desarrollo de la enfermedad en estudio, no así Xerostomía.

A escala mundial hay interés en la búsqueda de nuevos medicamentos antibacterianos para tratamientos de enfermedades infecciosas. Ante la comprobada resistencia y recurrencia de Candida albicans, Staphylococcus aureus y otras bacterias, se busca encontrar nuevos productos para el tratamiento contra estos microorganismos. El objetivo de este trabajo fue hallar la acción antifúngica del aceite de la cáscara de la nuez del Anacardium occidentale sobre Candida albicans y la actividad antibacteriana sobre Staphylococcus aureus por lo que se trabajó con cepas de la American Type Culture Collection y con cepas de pacientes portadores de VIH y de prótesis bucal. Se usó como control positivo a la Nistatina para C. albicans y Clorhexidina 2 % para S. aureus. Cada prueba se realizó por quintuplicado. Los resultados mostraron que el aceite del Anacardium occidentale no tiene actividad antifúngica, pero sí demostró tener gran actividad antibacteriana contra S. aureus, mucho mayor que la mostrada por la clorhexidina.

One of the major aims of this thesis was to identify novel regulators that drive C. albicans adaptation during growth under different nutrient and temperature conditions. The classical stress response cascades have been previously characterised under standardized, but physiologically irrelevant growth conditions (YPD at 30°C). In this study these pathways and other regulators were examined under more physiologically relevant inputs because metabolic plasticity and thermo-tolerance have been shown to affect stress adaptation (Arguelles et al., 1999; Brown et al., 2014; Cowen, 2009; Diezmann et al., 2014). In this study, we characterized 18.5% of the functional C. albicans ORFeome under 144 different stress conditions by employing a standardized system of robotic screening (Chapter 3). These screens highlighted extensive carbon and temperature-conditional regulators in C. albicans. We identified carbon-conditional contributions of the transcriptional regulators Sfp1 and Rtg3 to stress adaptation in this pathogenic fungus (Chapter 4). Sfp1 was found to regulate the expression of key stress regulators during growth on glucose, whereas Rtg3 induced the expression of these stress genes during growth on lactate. Our screens also revealed a distinct set of transcription factors, Hap43, Swi4, Sfp1, Cap1 and Zcf31, that control regulators of cell wall integrity and that promote antifungal drug resistance in a temperature dependent and yet Hsp90- independent manner. The screens also provided new information about a relatively obscure group of transcriptional regulators in C. albicans; the zinc cluster proteins with focus on Zcf3 and Zcf18 which we further pursued with RNA-sequencing to establish them as modulators of cell cycle, stress resistance and virulence in C. albicans. Lastly, our screens reveal a network of regulators that are homologous to human oncogenes and control fungal growth via modulation of TOR signaling. In conclusion, this thesis has revealed many novel targets for possible antifungal drug development and highlighted the extensive and intricate cross-talk between stress response modules facilitated by physiologically relevant nutrient sources and ambient temperatures.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Memoria para optar al Titulo Profesional de Médico Veterinario / Candida albicans es una levadura comensal que forma parte de la microbiota de las mucosas en el hombre, sin embargo es también un patógeno oportunista, responsable de infecciones invasoras, principalmente nosocomiales endógenas, las que presentan una alta morbiletalidad. Debido a que el estado sexual no es conocido en C. albicans, se espera que su estructura poblacional sea primariamente clonal, por esta razón un individuo puede portar clones de una cepa colonizante, en distintos lugares anatómicos, los que bajo ciertas condiciones, principalmente del hospedero, pueden producir infección. En Chile no existen estudios que revelen la relación de origen entre cepas colonizantes e infectantes, por lo tanto, resulta relevante profundizar el conocimiento sobre la epidemiología de la infección por C. albicans. El objetivo de este trabajo fue analizar genéticamente cepas patógenas de C. albicans, aisladas de hemocultivo, y cepas comensales provenientes de sitios colonizados de un mismo paciente con diagnóstico comprobado de candidemia, con el fin de determinar la relación de origen de estas cepas. Para esto, se analizaron molecularmente 35 cepas, identificadas previo al estudio como C. albicans, provenientes de ocho pacientes internados en Unidades de Pacientes Críticos del Hospital Clínico de la Universidad de Chile. De estas 35 cepas, cuatro se identificaron posteriormente como C. dubliniensis y provenían de un mismo paciente. Las cepas de C. albicans y C. dubliniensis fueron genotipificadas inicialmente mediante el método de Amplificación Aleatoria del ADN Polimórfico (RAPD), utilizando cinco partidores con actividad discriminatoria intraespecie. Se determinó el grado de similitud entre cepas mediante Coeficiente de Dice y se construyó un dendrograma, mediante el método UPGMA (Unweighted Pair Group Method Whit Arithmetic Averages). Los resultados mostraron que casi en la totalidad de los pacientes, los aislados propios de cada uno fueron agrupados en “cluster” únicos con una alta similitud genética (SAB ≥ 0.9), demostrando el origen clonal de los aislados y, en algunos casos, la existencia de fenómenos microevolutivos y reemplazo de cepas. 2 Posteriormente se analizaron las cepas de C. albicans a través de la Electroforesis en Gel con Gradiente Desnaturante (DGGE), mediante la amplificación de la porción final del 18S, el ITS1, el 5.8S y parte del ITS2 del ADN ribosomal. Los resultados del DGGE mostraron que las bandas pertenecientes a cada uno de los aislados en estudio migraron a la misma altura, sin conseguir evidenciar las diferencias genéticas existentes entre aislados de un mismo paciente y entre pacientes. El análisis estadístico, mediante el Test de McNemar, permitió establecer que el método de RAPD presenta un mayor rendimiento que el DGGE para la genotipificación de cepas de Candida spp. Finalmente, al comparar ambos métodos de genotipificación, sólo el RAPD pudo determinar que las cepas patógenas y comensales de C. albicans, provenientes de un mismo paciente con candidemia, están en su gran mayoría, relacionadas clonalmente.