Raman microspectroscopy enables one to acquire spectra of Raman scattering with a spatial resolution in the order of a few μm3 and thus to study the natural composition of biological objects such as tissues, single cells and cellular organelles in a non-invasive way. In this work, we used Raman microspectroscopy to investigate vacuoles of the opportunistic human yeast pathogen Candida albicans. Large sets of Raman spectra of vacuoles were collected based on different cultivation protocols. The sets of the spectra were evaluated using the multivariate statistical method of singular value decomposition. Based on the spectral analysis, we characterized the chemical composition of the vacuoles. We found out that the vacuoles of cells cultured differently or in different media vary particularly in the concentration of polyphosphate, represented in the spectra by the peak near 1155 cm-1 . Interestingly, the wavenumber position of the polyphosphate peak may also be shifted by several cm-1 . We studied these shifts in vitro with sodium hexametaphosphate as a model of vacuolar polyphosphate. Based on these experiments, we suggest that the peak position is significantly influenced by the concentration of divalent cations.

Candidate: Nikola Kollárová Title of diploma thesis: Detection of Sap2 in the secretome of Candida albicans cell wall and secretory mutants Charles University, Faculty of Pharmacy in Hradec Králové, Department of Biological and Medicinal Sciences Complutense University of Madrid, Faculty of Pharmacy, Department of Microbiology II Study program: Pharmacy Backgound: The aim of this diploma thesis was to search for C. albicans proteins involved in the secretion of the secreted aspartyl proteinase 2 enzyme (Sap2) evaluating the ability to degrade BSA (bovine serum albumin) as a source of nitrogen in several cell wall and secretory mutants of C. albicans. The work was carried out at the Department of Microbiology II, Faculty of Pharmacy, Complutense University of Madrid. Methods: The supernatant samples of several Candida albicans mutants were tested by SDS-PAGE electrophoresis and stained. Bands corresponding to BSA were observed and compared to controls. The other method was counted with 96-well plate. Results: The correlation between optical density and degradation of BSA was observed. Some mutants with disability to degrade BSA were found in a pilot screening of the ability to degrade BSA using 96-well plate method. That fact was confirmed by SDS-PAGE electrophoresis. C. albicans mutants showing...

Orientador: Maria Virginia Scarpa / Coorientador: Marcelo Gonzaga de Freitas Araújo / Banca : Luis Roberto Basso Junior / Banca : Leila Aparecida Chiavacci / Resumo: A curcumina é o constituinte majoritário encontrado no rizoma da planta Curcuma Longa L., e apresenta diversas atividades farmacológicas. Diversos estudos comprovaram que a curcumina possui excelente atividade antifúngica, sendo um princípio ativo natural promissor para o tratamento de doenças fúngicas. O objetivo específico deste trabalho foi o desenvolvimento de formulações contendo curcumina que possam ter ação contra a candidíase vulvovaginal (CVV). Para a análise estrutural das formulações foram utilizadas as técnicas de microscopia de luz polarizada, espalhamento de raios-X à baixo ângulos, difração de raios-X e comportamento reológico, sendo comprovado que os sistemas desenvolvidos apresentam características adequadas e satisfatórias para a aplicação pretendida e com propriedades mucoadesivas. O método analítico proposto para quantificação da curcumina foi revalidado e utilizado nos ensaios de estabilidade e liberação in vitro. As formulações desenvolvidas foram submetidas ao estudo de estabilidade preliminar, com o objetivo de garantir a segurança e eficácia das mesmas, e foi comprovada a estabilidade das formulações desenvolvidas. Posteriormente foram feitos os testes de liberação in vitro, para verificar a capacidade da curcumina de ser liberada a partir da formulação e exercer sua atividade farmacológica. O sistema emulsionado e o pseudoternário apresentaram taxas de liberação de 0,48% e 0,78% em 12 horas. A atividade farmacológica in vitro deste composto ativo, de... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Curcumin is the main active compound found in rhizome of Curcuma Longa L. plant and has several pharmacological activities. Several studies have shown that curcumin has excellent antifungal activity and it is a promising natural active compound for treatment of fungal diseases. The research objective was formulations development containing curcumin that may have action against vulvovaginal candidiasis (VVC). For formulations structural analysis were used techniques as polarized light microscopy, small angle X-ray scattering, X-ray diffraction and rheological behavior, and the developed systems has presented adequate and satisfactory characteristics according with research objective and with mucoadhesive properties. The proposed analytical method for curcumin quantification was revalidated, then used in stability study and in vitro release assays. The formulations were submitted to a preliminary stability study to guarantee its safety and efficacy, this way stability was proven. Subsequently were performed in vitro release tests to verify the curcumin ability of release from formulations and to exert its pharmacological activity. The emulsified and pseudoternary systems presented release rates of 0.48% and 0.78% in 12 hours. With the in vitro pharmacological activity test was determined the minimal inhibitory concentration against yeast Candida albicans and also the effective concentration to be incorporated in the systems. After these tests was performed in vivo anti-VVC acti... (Complete abstract click electronic access below) / Mestre

Orientador: Marlus Chorilli / Coorientador: Taís Maria Bauab / Banca: Hérida Regina Nunes Salgado / Banca: Marilisa Guimarães Lara / Resumo: A candidíase vulvovaginal (CVV) é uma infecção fúngica causada principalmente pela espécie Candida albicans, sendo considerada a mais prevalente em mulheres com idade fértil, acometendo cerca de 80-85% dos indivíduos ao menos uma vez na vida. Um crescente interesse tem sido observado na utilização de compostos de origem natural, como a curcumina, de forma a atuar contra este microrganismo. Todavia, algumas de suas propriedades físico-químicas, como baixa solubilidade aquosa, dificultam a sua utilização na terapêutica. Dessa forma, a sua incorporação em sistemas líquido-cristalinos mucoadesivos (SLCM) objetivando administração vaginal pode ser uma estratégia eficiente de tratamento, de forma a potencializar a ação do fármaco, bem como oferecer maior permanência da formulação no local de ação. O objetivo deste trabalho foi desenvolver SLCM constituídos por ácido oleico e ergosterol (5:1) como fase oleosa, ácido cetílico etoxilado e propoxilado (Procetyl® AWS) como tensoativo e dispersão polimérica de quitosana (1%) como fase aquosa, acrescidos de curcumina, e caracterizá-los empregando microscopia de luz polarizada, espalhamento de raios-X de baixo ângulo (SAXS), reologia, análise de textura e mucoadesão in vitro. Posteriormente, avaliou-se a ação antimicrobiana in vitro e in vivo do sistema contra cepas de C. albicans. Pelos ensaios de microscopia de luz polarizada as formulações apresentaram estruturas semelhantes à cruz de malta e campo escuro, características de fase lamela... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Vulvovaginal candidiasis (VVC) is a fungal infection mainly caused by the Candida albicans species. The disease is being considered the most prevalent in women of childbearing age, affecting approximately 80-85% of individuals at least once in life. An increasing interest has been observed in the use of compounds of natural origin, such as curcumin, in order to act against this microorganism. However, some of its physicochemical properties, such as low aqueous solubility, make it difficult to use in therapeutics. Thus, its incorporation into mucoadhesive liquid-crystalline systems (MLCS), with vaginal administration, can be an efficient treatment strategy in order to potentiate the action of the drug, as well as to offer a longer permanence of the formulation at the site of action. The aim of this work was to develop mucoadhesive liquid-crystalline systems consisting of oleic acid and ergosterol (5: 1) as oily phase, ethoxylated and propoxylated cetylic acid (Procetyl® AWS) as a surfactant and polymer dispersion of chitosan (1%) as aqueous phase, plus curcumin and characterize them using polarized light microscopy, low-angle X-ray scattering (SAXS), rheology, texture analysis and in vitro mucoadhesion. Subsequently, the antimicrobial action in vitro and in vivo of the system against C. albicans strains was evaluated. By polarized light microscopy, the formulations presented similar structures to the cross of malt and dark field, characteristics of lamellar phase and microemulsions, respectively, the results were confirmed by low-angle X-ray scattering. In vitro mucoadhesion assays demonstrated an increase in the mucoadhesive strength of the formulations in the presence of increasing concentrations of artificial vaginal mucus (AVM), corroborating rheology results, which showed high viscosity and elasticity of the formulations in the presence of AVM... (Complete abstract click electronic access below) / Mestre

The fungal pathogen Candida albicans and the opportunistic bacterial pathogens Enterobacter aerogenes and Enterobacter cloacae are common sources of human disease. The colonization of proximal anatomical locations by these pathogens suggests that interspecies polymicrobial interactions between Candida albicans and Enterobacter species occur. In order to understand mechanisms of diseases caused by these pathogens and to further the study of disease prevention, analyzation of their combined activities was conducted in this study. Changes in fungal morphology, cellular viability, and colony density were investigated using fungal and bacterial co-cultures. The effects of the Candida secreted quorum sensing molecule farnesol on Enterobacter aerogenes and Enterobacter cloacae was studied to observe changes in Enterobacter viability and colony density. The effects of the presence of Enterobacter species on Candida albicans was studied by observing changes in Candida morphology and colony density. The mutant strain of Candida albicans AlS6-/- was also cultured with Enterobacter to determine if the presence of the ALS6 surface glycoprotein gene affected Candida viability and colony density in the presence of Enterobacter species. Statistically significant decreases were observed in all studied metrics between experimental and control groups. This indicated that the interactions observed between Candida albicans and Enterobacter species represent reciprocal inhibitions of cellular functionalities. As Candida albicans is the primary cause of human fungal infections and Enterobacter species are common causes of opportunistic infections, the study of polymicrobial interactions between Candida and Enterobacter species as conducted in this study is important to furthering efforts of human disease inhibition.

Candida albicans is the most common human fungal pathogen. The relationship between C. albicans and Enterobacter bacteria have yet to be explored. The hypothesis of this study is that C. albicans and both E. aerogenes and E. cloacae have a positive relationship and work together to infect the host. In this study, the physical cell-to-cell interaction, molecular components of said interaction, and the impact of the interaction on a live organism were explored. Results indicate that Enterobacter adheres to C. albicans and inhibits growth with unidentified secreted molecules. Als1p has potential involvement in the attachment of E. cloacae to C. albicans. Out of 480 E. cloacae mutants, 6 showed reductions in attachment to C. albicans. The presence of C. albicans in C. elegans may lead to less Enterobacter colonization. Future work involving this interaction should strive to identify the Enterobacter secreted molecules and genes necessary for their production.

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED, APS-I) is exceptionally common in Finland. Most patients have chronic oral candidiasis since childhood. Thus, most patients receive repeated courses of antifungals throughout their life. Eleven of our patients (31.4%) have become colonized with Candida albicans with decreased sensitivity to fluconazole. A total of 43 isolates of C. albicans from 23 APECED patients isolated during the years 1994 to 2004 were divided into 2 groups: fluconazole-susceptible dose-dependent (MIC, 16-32 μg/mL, 18 isolates) and fluconazole-susceptible (MIC ≤8 μg/mL, 25 isolates) groups. Antifungal activity of amphotericin B, echinocandins, and azoles was determined by the Clinical and Laboratory Standards Institute M27-A2 methodology. All isolates were highly susceptible to amphotericin B and echinocandins. Posaconazole and voriconazole were active against all isolates. Our data suggest that topical amphotericin B could continue to be a safe and active drug for daily administration for APECED patients. Posaconazole, voriconazole, and echinocandins may be useful in some complicated cases.

Objectives: Hormones and cytokines are known to act as regulatory messengers between the neuroendocrine and immune systems. The innate immune system identifies infectious agents by means of pattern-recognition receptors. These receptors recognize pathogen-specific macromolecules called pathogen-associated molecular patterns. Fungal cell wall glucans nonspecifically stimulate various aspects of innate immunity via interaction with membrane receptors on immune-competent cells. Glucans are also released into the systemic circulation of patients with fungal infections. Recent evidence confirms the existence of glucan-specific receptors on cells outside the immune system. We hypothesized that glucans may directly interact with pituitary cells as an early signaling event in fungal infections. Methods: We characterized the receptor-mediated interaction of glucan derived from Candida albicans with pituitary cells using surface plasmon resonance. Prolactin levels were assayed by commercial ELISA. TLR2, TLR4 and CD14 mRNA levels were assessed by RT-PCR. Results: A single glucan-specific binding site was identified on rodent somatomammotroph (KD = 3.9) μM) and human folliculostellate cell (KD = 3.6) μM) membranes. Coincubation of glucan with somatomammotroph cells for 72 h significantly (p < 0.01) increased prolactin accumulation by 56-62% over that observed in cells treated with media alone. Glucan also increased TLR4 and CD14 gene expression in human folliculostellate cells. Conclusions: Pituitary cells directly recognize and respond to fungal cell wall glucans resulting in stimulation of pituitary cell TLR4 and CD14 gene expression. In addition, glucan stimulates secretion of prolactin, a hormone that plays an important role in the response to infection.

Philosophiae Doctor - PhD / The Black soldier fly (BSF) is an environmentally friendly and sustainable insect utilised in the decomposition of organic waste. This is due to its voracious consumption capability, disruptive functions and economic importance. The sustained global increase in commercial BSF farming has resulted in an expanded waste generation from its carcases to which beneficial uses ought to be developed. This study focused on the beneficial use of the generated waste by extracting chitosan from waste pupae and commercially reared BSF adult carcases. The study also considered the conversion of the extracted chitosan to nanofibres and nanoparticles for application in adsorption of inorganic Pb2+ or Cd2+ and antimicrobial studies, respectively. To achieve the aim of this study, the optimal extraction conditions of chitin and chitosan from both pupal exuviae and adult BSF waste materials were attained after a series of experiments. The extraction process involved three stages which were demineralisation, deproteination and deacetylation. The extracted adult and pupal chitin and chitosan were characterised using Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), X-ray diffraction studies (XRD), high resolution scanning electron microscopy (HRSEM) and solid-state carbon nuclear magnetic resonance spectroscopy (13C NMR). Additionally, the adult (ACH20_9) and pupal (PCH21_9) chitosan samples, due to their solubility, were further characterised to determine their molecular weight, fat and water binding capacities, solubility and ash contents. / 2021-09-30

El presente trabajo investigo la actividad antifúngica in vitro de doce extractos etanolicos correspondientes a diez plantas medicinales peruanas; Annona cherimolia Mill. (hojas), Annona muricata L. (corteza y hojas), Bidens pilosa L. (partes aéreas), Hypericum laricifolium L. (partes aéreas), Juglans neotropica Diels (corteza), Piper spp. (hojas), Plantago major L. (hojas), Psidium guajava L. (hojas), Schinus molle L. (corteza y hojas) y Spartium junceum L. (planta entera). Las especies fueron recolectadas en el departamento de Amazonas, excepto Schinus molle L. (Apurímac) y Annona muricata L. (Lima). La actividad antifúngica se evaluo mediante los métodos de difusión en agar y dilución en agar para la determinación de la concentración mínima inhibitoria (CMI). Los microorganismos de prueba utilizados fueron las levaduras Candida albicans ATCC 10231 y Candida albicans cepa clínica, así como, el hongo filamentoso Aspergillus niger ATCC 16404; las cepas fueron proporcionadas por la Cátedra de Microbiología de la Facultad de Farmacia y Bioquímica de la Universidad Nacional Mayor de San Marcos. De doce extractos investigados, seis presentaron actividad antifúngica consistente con un diámetro de halos de inhibición ≥18mm (Prueba de Difusión en agar) frente a Candida albicans ATCC 10231. Ningún extracto mostró actividad consistente frente a la cepa clínica de Candida albicans y Aspergillus niger ATCC 16404. La CMI de los extractos que presentaron actividad consistente frente a Candida albicans ATCC 10231, fue de 250 µg/mL para Hypericum laricifolium L., Juglans neotropica Diels, Psidium guajava L. y Schinus molle L. (un extracto de corteza y uno de hojas) y de 500µg/mL para Piper spp. No se determino la CMI de los extractos (Juglans neotropica Diels y Psidium guajava L.) que presentaron halos frente al Aspergillus niger ATCC 16404 por considerarlos sin actividad significante (<18mm.). Los antifúngicos Nistatina y Fluconazol fueron incluidos en el estudio como controles positivos. -- Palabras clave: Actividad antifúngica, plantas medicinales, plantas del Perú, Amazonas, concentración mínima inhibitoria, Candida albicans, Aspergillus niger. / -- The present work investigated the antifungal activities in vitro of twelve ethanolic extracts corresponding to ten peruvian medicinal plants; Annona cherimolia Mill. (leaves), Annona muricata L. (bark and leaves), Bidens pilosa L. (aerial parts), Hypericum laricifolium L. (aerial parts), Juglans neotropica Diels (bark), Piper spp. (leaves), Plantago major L. (leaves), Psidium guajava L. (leaves), Schinus molle L. (bark and leaves) and Spartium junceum L. (whole plant). The plants were collected in the department of Amazonas, except Schinus molle L (Apurímac) and Annona muricata L. (Lima). The antifungal activities were determinated by the methods of agar diffusion and agar dilution for the determination of the Minimal Inhibitory Concentration (MIC). The used microorganisms of test were the yeasts Candida albicans ATCC 10231 and Candida albicans clinical isolate, as well as, the filamentous fungus Aspergillus niger ATCC 16404; the microorganism were provided by the Chair of Microbiology of Faculty of Pharmacy and Biochemistry of the Universidad Nacional Mayor de San Marcos. Of twelve investigated extracts, six presented significant antifungal activity with a diameter of inhibition haloes ³18mm (Agar Diffusion) against Candida albicans ATCC 10231. No extract showed significant activity against to Candida albicans clinical isolate and Aspergillus niger ATCC 16404. The MIC of the extracts that presented significant activity against Candida albicans ATCC 10231, were of 250µg/mL for Hypericum laricifolium L., Juglans neotropica Diels, Psidium guajava L. and Schinus molle L.(bark and leaf extracts) and of 500µg/mL for Piper spp. We do not determine the MIC of the extracts (Juglans neotropica Diels and Psidium guajava L.) that presented haloes against Aspergillus niger ATCC 16404 for considering them without significant activity (<18mm.). The antifungal agents Nistatin and Fluconazole were included in the study as positive controls. -- Key words: Antifungal activity, medicinal plants, Peruvian plants, Amazonas, minimal inhibitory concentration, Candida albicans, Aspergillus niger. / Tesis