A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science in Medicine. / Introduction Despite the availability of several antifungal drugs, Candida infections remain a major health threat worldwide. The Candida infections problem has been amplified by the emergence of multidrug resistant Candida species towards the conventional antifungal drugs. In addition, activation of antioxidant defense system by Candida species has been known to be forefront mechanism to escape drug toxicity.This indicates an urgent need for the development of new therapeutic strategies and antifungal drugs. Natural products have served for centuries for the treatment of infectious diseases and are among the major sources for finding new antifungal drugs. Berberine (BER), an isoquinoline alkaloid found in a variety of plant species, has been shown to possess multiple biological and pharmacological properties including antimicrobial activity against C. albicans and other Candida species. However, the mechanism of action exerted by BER and its effect on Candida cells is not yet fully elucidated. Therefore, this study was conducted to evaluate the role of antioxidant enzymes in the susceptibility to fluconazole (FLC) in C. albicans. Another aspect was to determine the effect of BER on growth, antioxidant enzymes and their gene expression in C. albicans. Materials and methods Candida albicans clinical isolates (10 FLC susceptible and 10 FLC resistant) and one ATCC strain were obtained from the Department of Clinical Microbiology and Infectious Diseases, University of the Witwatersrand. Species identification was confirmed using API 20C AUX. Antifungal susceptibility was determined following CLSI M27-A3 guidelines. Gene expression of SOD1, SOD2, GPx2, GLR1, GTT11, and CAT1 in untreated and BER treated C. albicans cells was measured by RT-qPCR. The activity level of the corresponding enzymes in the presence of BER was determined using a spectrophotometer. Results Gene expression analysis showed an increase in mRNA expression level of SOD1, SOD2, GPx2, GLR1 and GTT11 genes in FLC resistant isolates than in the susceptible group. The most significantly expressed gene was SOD1 with 50.69-fold increase. The other genes showed moderate increase in the expression with fold change ranging from 1.2 to 4.2. The susceptibility test showed MICs ranging from 125 to 500 μg/ml with a significant difference in the activity of BER between FLC susceptible and resistant C. albicans. BER treatment induced upregulation in the mRNA expression and enzymatic activities of major antioxidants. In FLC resistant C. albicans, treatment with ½ MIC value of BER caused downregulation of the targeted antioxidant genes indicating that BER at this concentration induced an intense oxidative stress, therefore, surpassing the antioxidant capacity of the cells. Conclusion The findings in this study showed that drug resistance is not only caused by mutations in a particular gene but could also arise from proteomic modulations. The study . The study also demonstrated that C. albicans activates several antioxidant enzymes that form an integral component of the cell’s response against oxidative stress. Candida albicans showed efficient antioxidant response at lower concentrations of BER. However, BER at ½ MIC value induced robust oxidative stress, especially in FLC resistant C. albicans, surpassing the antioxidant capacity of the cells. This demonstrates that BER at sub-inhibitory concentrations is able to render C. albicans avirulent by suppressing its antioxidant defense response without compromising cell viability of the fungi. Therefore, BER has a potential to be developed into BER has a potential to be developed into a therapeutic agent a therapeutic agent for for the the treatment oftreatment of C. albicansC. albicans infections and other pathogenic fungi to infections and other pathogenic fungi to overcome drug resistance. overcome drug resistance. / E.K. 2019

The fungal cell wall serves as the interface between the organism and its environment. Complex carbohydrates are a major component of the cell wall, , glucan, mannan and chitin. β-Glucan is a pathogen associated molecular pattern (PAMP) composed of β-(1 → 3,1 → 6)-linked glucopyranosyl repeat units. This PAMP plays a key role in fungal structural integrity and immune recognition. Glycogen is an α-(1 → 4,1 → 6)-linked glucan that is an intracellular energy storage carbohydrate. We observed that glycogen was co-extracted during the isolation of β-glucan from SC5314. We hypothesized that glucan and glycogen may form a macromolecular species that links intracellular glycogen with cell wall β-(1 → 3,1 → 6)-glucan. To test this hypothesis, we examined glucan-glycogen extracts by multi-dimensional NMR to ascertain if glycogen and β-glucan were interconnected. H NMR analyses confirmed the presence of glycogen and β-glucan in the macromolecule. Diffusion Ordered SpectroscopY (DOSY) confirmed that the β-glucan and glycogen co-diffuse, which indicates a linkage between the two polymers. We determined that the linkage is not via peptides and/or small proteins. Our data indicate that glycogen is covalently linked to β-(1 → 3,1 → 6) glucan via the β -(1 → 6)-linked side chain. We also found that the glucan-glycogen complex was present in , and , but was not present in or hyphal glucan. These data demonstrate that glucan and glycogen form a novel macromolecular complex in the cell wall of and other species This new and unique structure expands our understanding of the cell wall in species.

Strains of pathogens are typically described as virulent or non-virulent. However, in the majority of pathogens, strains often vary continuously and quantitatively in their virulence and pathogencity. Biofilm formation is one of the recently recognized virulence factors in many human pathogens and little is known about the variation and evolution of biofilms among natural strains. In this study, I examined quantitative variation of biofilms among natural strains of the human pathogenic yeast Candida albicans. A total of 115 natural strains of C. albicans from three sources (vaginal, oral and environmental) were quantified by two mebods: (i) the XTT tetrazolium reduction assay, and (ii) optical density following staining by crystal violet dye. Mature biofilm was confirmed by observation using confocal laser scanning microscopy. My analyses indicated that strains from each of the three scurces varied widely in biofilm formation abilities and that biofilm formation ability was positively correlated to cell surface hydrophobicity (CSH). For each strain, multilocus genotypes were determined by PCR-RFLP, my comparative genotype and biofilm analyses denonstrated that natural clones and clonal lineages of C. albicans exhibited extensive quantitative variation for biofilm formation. I also examined potential interactions among strains within C. albicans and between different Candida species. My preliminary results suggest significant variation and complex patterns of strains or species interaction during Candida biofilm development. / Thesis / Master of Science (MS)

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

Thesis (M.A.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / Gandida albicans strain 105 from a normal human and strain 582 (from the American type Culture Collection) were used for studying the effect in vitro of pH, various amounts of carbon dioxide, nitrogen, and composition of media on filamentation in this yeast-like organism. The yeast phase of the organism was maintained on a glucose, glycine, yeast extract (GGY) medium (1%; 1%; 0.5%) at 37°C. The experiments were conducted on both solid and liquid media. All cultures were incubated at 37°C. for 48 hours. The two strains of c. albicans, although similar to one another in their yeast forms, behaved differently toward the environmental conditions used; strain 582 responded more readily to the factors inducing filament formation than did strain 105. Increasing the pH above 6.5 to 7.0, 7.5 and 8.0 induced maximum filamentation in strain 582, whereas no filaments were produced by strain 105. All the aerobic cultures on solid GGY medium showed alkalinity and were positive for ammonia at the end of the incubation period. In liquid media, no alkalinity was observed at any pH values. Presence of 75% carbon dioxide in the atmosphere increased filamentation in strain 582 to a maximum degree, and induced mycelial formation in strain 105. With 94% or 95% carbon dioxide, growth and filamentation decreased in both strains. None of the CO2 cultures showed alkalinity at the end of the incubation period. Moreover, all the CO2 cultures were negative for ammonia. Growth under nitrogen (9J%) was less than that of the aerobic cultures. However, colonies appeared larger in size. Nitrogen stimulated filamentation in strain 105 only at a pH of 8.0, whereas strain 582 formed a maximum amount of filaments at pH values of 7.0 to 8.0. All the solid cultures under nitrogen showed alkalinity, while the liquid cultures were acid at all pH values. The occurrence of deamination in a medium without glucose in both strains of C. albicans showed that this organism was able to use glycine its source of both nitrogen and carbon. However, only a sparse growth was obtained in a medium lacking glucose. Strain 105 did not form filaments in such a medium, while strain 582 did so. Since more filaments were produced by the latter strain when a fresh subculture on a GGY medium was transferred to a medium without glucose, it was concluded that possibly glucose is required for both growth and filamentation. Comparative studies of the effect of a medium containing mannose with a glucose medium showed the two sugars behaved similarly with regard to fermentation and filament induction in both strains or c. albicans. Under conditions where glucose induced filamentation (e.g., with C02 or N2), mannose also induced filamentation. The decreased growth in the presence of oleic or stearic acid in a concentration of 40 micrograms per liter was attributed to the toxic effect of the fatty acids. Moreover, it was noted that the two acids had different effects on filamentation in the two strains. Oleic acid in a solid GGY medium induced hyphal formation in strain 105 only under nitrogen; without glucose, oleic acid did not bring about filamentation under any of the atmospheric conditions tested. In liquid media, oleic acid induced filamentation for strain 105 only when glucose was omitted. With strain 562, oleic acid promoted filamentation in both liquid and solid media with or without glucose, except for solid cultures incubated under nitrogen in the absence or glucose. Stearic acid did not stimulate filamentation in strain 105 under any conditions, but did increase hypha! formation in strain 582. In the presence of stearic acid, maximum filamentation occurred in aerobic cultures wnen glucose was absent. Although maximum filamentation occurred with an increase in the pH of the medium under aerobic conditions, in the presence of 75% C02, under nitrogen or in the presence of stearic acid in a medium without glucose, yeast cells were also present, indicating that this Y to f transformation was not complete. / 2999-01-01

Durant les 20 dernières années, les infections fongiques ont augmenté de façon considérable et causent un défi dans le domaine médical. C. albicans est responsable de 20 % à 75 % de la majorité des candidoses. La candidose buccale se produit à plus de 80% chez les patients infectés par le VIH, et C. albicans a pu être identifié chez 78-100 % des porteurs de prothèses dentaires souffrant de stomatite prothétique. Lors de traitement, les antifongiques ont une efficacité limitée. Dans le but de trouver un nouvel antifongique qui pourrait résoudre les problèmes de résistance de C. albicans aux antifongiques disponibles actuellement, nous avons évalué in vitro l'effet du famesol sur la croissance et la transformation de C. albicans ainsi que son innocuité sur les cellules gingivales humaines. Nous avons d'abord étudié l'effet du famesol à différentes concentrations (0 à 300 uM) sur la croissance et la transformation de C. albicans. Pour tester l'innocuité du famesol, nous avons utilisé des cellules épithéliales et des fibroblastes isolés de biopsies de tissus palatins. L'effet du famesol sur la croissance et la transformation de C. albicans a été analysé par numération cellulaire. L'innocuité a été étudiée par des techniques de microscopie optique et par numération cellulaire. Avec les différentes cultures cellulaires qu'on a réalisées, nous avons montré que le famesol réduit la croissance de C. albicans et inhibe sa transformation de la forme blastospore à la forme hyphe. Les études de l'innocuité du famesol sur les cellules gingivales humaines ont montré que le famesol était nocif pour les fibroblastes (70 uM) mais les cellules épithéliales étaient plus résistantes (150 uM). Nos travaux montrent aussi que le famesol et les cellules gingivales humaines ont des effets synergiques contre la transformation de C. albicans. Ces études suggèrent que le famesol pourrait être utilisé comme un agent antifongique.

Background: Plastic stents used for the treatment of biliary obstruction will become occluded over time due to microbial colonization and formation of biofilms. Treatment of stent-associated cholangitis is often not effective because of inappropriate use of antimicrobial agents or antimicrobial resistance. We aimed to assess the current bacterial and fungal etiology of stentassociated biofilms, with particular emphasis on antimicrobial resistance. Methods: Patients with biliary strictures requiring endoscopic stent placement were prospectively enrolled. After the retrieval of stents, biofilms were disrupted by sonication, microorganisms were cultured, and isolates were identified by matrix-associated laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and/or biochemical typing. Finally, minimum inhibitory concentrations (MICs) were determined for various antimicrobial agents. Selected stents were further analyzed by fluorescence in situ hybridization (FISH). Results: Among 120 patients (62.5% males, median age 64 years) with biliary strictures (35% malignant, 65% benign), 113 double pigtail polyurethane and 100 straight polyethylene stents were analyzed after a median indwelling time of 63 days (range, 1–1274 days). The stent occlusion rate was 11.5%and 13%, respectively, being associated with a significantly increased risk of cholangitis (38.5% vs. 9.1%, P<0.001). Ninety-five different bacterial and 13 fungal species were detected; polymicrobial colonization predominated (95.8% vs. 4.2%, P<0.001). Enterococci (79.3%), Enterobacteriaceae (73.7%), and Candida spp. (55.9%) were the leading pathogens. Candida species were more frequent in patients previously receiving prolonged antibiotic therapy (63% vs. 46.7%, P = 0.023). Vancomycinresistant enterococci accounted for 13.7%, extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae with co-resistance to ciprofloxacin accounted for 13.9%, and azole-resistant Candida spp. accounted for 32.9% of the respective isolates. Conclusions: Enterococci and Candida species play an important role in the microbial colonization of biliary stents. Therefore, empirical antimicrobial treatment of stent-associated cholangitis should be guided toward enterococci, Enterobacteriaceae, streptococci, anaerobes, and Candida. To determine causative pathogens, an accurate microbiological analysis of the extracted stent(s) may be helpful.

O objetivo do presente estudo foi avaliar, in vitro, o efeito de diferentes tempos de pré-irradiação do fotossensibilizador na terapia fotodinâmica em biofilmes formados por Streptococcus mutans e Candida albicans, por meio da avaliação da carga microbiana. Os fatores em estudo foram: tempos de pré-irradiação do fotossensibilizador em 3 níveis (1, 2 ou 5 minutos). Para o controle do biofilme dentário cariogênico com aPDT foi utilizado o azul de metileno (0,01%) associado ao laser de diodo (&lambda;=660 nm). O digluconato de clorexidina (CHX a 0,12%) e a solução salina foram utilizados como controle positivo e negativo, respectivamente. O delineamento do estudo foi realizado em blocos completos e casualizados, sendo a amostra composta por 15 culturas de biofilmes de S. mutans, divididas aleatoriamente em 5 grupos e 15 culturas de C. albicans, também divididas em 5 grupos. O experimento foi realizado em triplicata (n=3) e as variáveis de resposta foram obtidas por meio de análise quantitativa da viabilidade bacteriana, expressa em unidades formadoras de colônia (UFC) por mm2 da área do espécime. Os dados obtidos foram analisados com o auxílio do teste one-way ANOVA e pós-teste de Tukey. Todas as análises foram efetuadas por meio do programa Graph Pad Prism 4.0, com nível de significância de 5%. Para o grupo de S. mutans, apenas a solução salina apresentou diferença estatisticamente significante quando comparada aos demais tratamentos (p<0.05), ou seja, o tratamento com aPDT, independentemente do tempo de irradiação aplicado, foi semelhante ao tratamento com CHX e ambos foram mais eficazes na redução do biofilme cariogênico, em comparação à solução salina. Para o grupo de C. albicans não houve diferença estatística entre os grupos (p>0.05). Portanto, pode-se concluir que o tratamento com aPDT diminuiu o número de UFCs de S. Mutans de forma semelhante à CHX, independentemente do tempo de pré-irradiação aplicado. Não foi possível constatar nenhum efeito desta terapia e dos diferentes tempos de pré-irradiação sobre o biofilme de C. albicans. Desta forma, o tempo de pré-irradiação de 1 minuto pode ser utilizado com o objetivo de reduzir a carga microbiana de S. Mutans. / The aim of the present study was to evaluate, in vitro, the effect of different pre-irradiation times of the photosensitizer in photodynamic therapy in biofilms formed by on by Streptococcus mutans and Candida albicans, through the evaluation of the microbial load. The factors under study were: times of pre-irradiation of the photosensitizer in 3 levels (1, 2 or 5 minutes). For the control of the cariogenic dental biofilm with aPDT, methylene blue (0.01%) was used in association with the diode laser (&lambda;=660 nm). Chlorhexidine digluconate (0.12% CHX) and saline were used as positive and negative controls, respectively. The study design was carried out in complete and randomized blocks. The sample consisted of 15 S. mutans biofilms cultures, randomly divided into 5 groups and 15 C. albicans cultures, also divided into 5 groups. The experiment was performed in triplicate (n = 3) and the response variables were obtained through quantitative analysis of bacterial viability, expressed in colony forming units (CFU) per mm2 of the specimen area. The data were analyzed with the aid of the ANOVA one-way test and Tukey\'s post-test. All analyzes were performed using the Graph Pad Prism 4.0 program, with a significance level of 5%. For the S. mutans group, only the saline solution presented a statistically significant difference when compared to the other treatments (p <0.05), that is, the treatment with aPDT, irrespective of the irradiation time applied, was similar to the treatment with CHX and both were more effective in reducing cariogenic biofilm compared to saline. For the group of C. albicans there was no statistical difference between the groups (p> 0.05). Therefore, it can be concluded that the treatment with aPDT reduced the number of CFUs of S. Mutans in a similar way to CHX, independently of the pre-irradiation time applied. No effect of this therapy or of the different pre-irradiation times on the C. albicans biofilm could be observed. In this way, the pre-irradiation time of 1 minute can be used to reduce the microbial load of S. mutans.

A incidência de infecções por fungos oportunistas na população de pacientes imunocomprometidos tem aumentado nos últimos anos, e estas são, principalmente, causadas por Candida albicans. Este patógeno oportunista pode crescer em diferentes formas, variando de levedura, pseudohifa e hifa e essa transição morfológica está associada com a virulência. Na transição de levedura para hifa, genes hifa-específicos são expressos e muitos deles codificam proteínas que possuem uma molécula de GPI (glicosilfosfatilinositol), mas a maioria (66%) das proteínas preditas ancoradas por GPI (PGAs), ainda possuem função desconhecida. Portanto, o objetivo deste trabalho, foi iniciar a caracterização funcional dos genes PGA13 e PGA58 através de análises in silico, da expressão diferencial dos RNAm sob condições ambientais diversas, e nos mutantes nulos TUP1 e EFG1, e a obtenção e caracterização fenotípica dos mutantes de PGA13: homozigoto nulo e de superexpressão. Os estudos realizados in silico indicam que PGA13 e PGA58 são reguladas pelos fatores transcricionais Efg1, Tec1 e Nrg1 e que possuem sítios potenciais de glicosilação. A análise transcricional desses genes mostra que ambos são regulados por Tup1 e que respondem aos estímulos NaCl (Cloreto de sódio), H2O2 (Peróxido de hidrogênio), etanol, cafeína, HCl (Ácido Clorídrico) e às condições hipo e hiperosmótica. Os mutantes nulos e de superexpressão de PGA13 e PGA58 foram obtidos e as linhagens que perderam o gene PGA13 tiveram um padrão de crescimento da colônia diferente da linhagem CAI-4 e foram mais sensíveis aos compostos SDS, Higromicina B e pH ácido, enquanto o mutante que superexpressava foi mais resistente. Estes resultados sugerem que Pga13 deve ter papel importante na parede celular, visto que a falta dela ocasionou maior sensibilidade a compostos que agridem a parede celular. / The incidence of opportunistic fungal infections in immunocompromised patients has increased in the last years. Candida albicans is the most commonly isolate in immunocompromised patients. C. albicans may grow in distinct morphologies: yeast, pseudohyphal and true hyphal. The switch between yeast and filamentous forms is strongly associated with virulence. During the transition, hyphal-specific genes are expressed and many of these genes encode glycophosphatidylinositol (GPI)- anchored proteins. The majority (66%) of predited GPI proteins has unknown functions. The purpose of this work was to functionally characterize the novel PGA13 and PGA58 genes. To accomplish this task, we have made in silico promoter analysis, mRNA differential expression analysis under some environmental conditions. We have also verified whether those genes are regulated by Tup1 and Efg1 transcriptional regulators. We have knocked out PGA13 and have done phenotypic analysis of the resulting PGA13 null mutant strain and a Pga13 overexpressing strain. The in silico promoter analysis indicates that PGA13 and PGA58 may be regulated by the transcriptional factors Efg1, Tec1 and Nrg1. PGA13 and PGA58 aminoacid sequence analysis revealed that they have potential glycosylation sites. The transcriptional analysis has shown that both genes are regulated by the morphogenesis negative regulator Tup1. PGA13 and PGA58 genes have differential expression in response to salt, hydrogen peroxide, ethanol, caffein, and acid stresses, and also to hypo and hyperosmotic shocks. The phenotypic analysis shows that the pga13/pga13 mutant was more sensitive to SDS, Hygromycin B and acid pH in a plate dilution sensitivity test. Interestingly, the overexpressing mutant strain was more resistant to the same compounds. Taken together these results suggest that PGA13 may have an important role in the cell wall since its absence leads to higher sensitivity to compounds that affect this organelle.

A Estomatite protética associada a Candida (EPC) acomete a mucosa bucal em contato com próteses removíveis e, clinicamente, caracteriza-se por eritema com diferentes graus de inflamação. Esta doença é considerada de etiologia multifatorial, isto é, uma associação de fatores como trauma, falta de higienização, uso contínuo da prótese, hipersensibilidade ao material usado na dentadura, diabetes, terapia imunossupressora e infecção por fungo, como diferentes espécies de Candida. Os principais fatores de virulência deste fungo são a formação de hifas, dimorfismo, alterações fenotípicas, aderência, persistência, sinergismo com as bactérias, interferências com o sistema de defesa do hospedeiro e a produção de enzimas hidrolíticas. Dentre as enzimas hidrolíticas, a proteinase aspartil secretada (SAP) é uma das mais importantes para a patogenia de C. albicans, sendo nociva para o tecido epitelial e para o sistema imune do hospedeiro. Não está totalmente compreendida a real penetração do fungo nos tecidos e sua correlação com a presença da SAP, na doença estomatite protética. Essa dificuldade de avaliação pode ser justificada pelas divergências intrínsecas e extrínsecas observadas em muitos aspectos, como diferentes costumes, hábitos sociais, estado emocional e fisiológico. A utilização de um modelo experimental em animais poderá minimizar essas divergências e fornecer condições mais padronizadas para o experimento. Neste trabalho, foram avaliadas, quantitativamente, a expressão gênica das enzimas SAP-2, SAP-5 e SAP-9, presentes no biofilme formado na superfície interna das placas acrílicas superiores de ratos e, microscopicamente, a invasão do fungo no tecido epitelial do palato. Para isso, foram selecionados 49 ratos Wistar, com 90 dias de vida, pesando em média 300g, os quais foram divididos em 3 grupos: Controle, Placa/Candida e Placa, acompanhados durante 2, 4 e 6 dias. Os resultados demostraram que, em 4 dias de uso da placa acrílica contaminada, houve, em alguns ratos, sinais clínicos de inflamação no palato duro; microscopicamente, hiperplasia epitelial, hiperqueratinização, invasão fúngica nas camadas superficiais do revestimento epitelial, microabscessos de Munro e hiperplasia papilar; e maior percentual de neutrófilos no Grupo Placa/Candida em relação aos Grupos Controle e Placa. Também no quarto dia de uso da placa acrílica superior, no Grupo Placa/Candida, o biofilme formado na sua superfície interna apresentou a mais alta expressão gênica relativa das enzimas SAP-2, SAP-5 e SAP-9 que os períodos de 2 e 6 dias de uso. Assim, a invasão fúngica no revestimento epitelial do palato duro pode estar correlacionada com a alta expressão de RNAm das SAPs -2, -5 e -9. / Denture stomatitis (D.S.) affects the oral mucosa in contact with removable dentures, and clinically characterized by erythema with varying degrees of inflammation. This disease is considered a multifactorial etiology, with a combination of factors such as trauma, lack of hygiene, continuous use of stents, hypersensitivity to the material used for dentures, diabetes, immunosuppressive therapy and fungal infection, such as different species of Candida. The main virulence factors of the fungus are the formation of hyphae, dimorphism, phenotypic changes, adherence, persistence, synergism with bacteria, interference with the host defense system and the production of hydrolytic proteins. Among the hydrolytic proteins, the secreted aspartyl proteinase (SAP) is one of the most important in the pathogenesis of C. albicans. SAP is harmful to both the epithelial tissue and to the host\'s immune system. It is not fully understood the real penetration of the fungus in the epithelium tissue and its correlation with the presence of SAP, in denture stomatitis. This difficulty in evaluation can be justified by the intrinsic and extrinsic differences observed in many aspects, different customs, social`s habits, emotional and physiological state. Using an experimental animal model may minimize these differences and provide more standardized conditions for the experiment. In the present work, the aim was to evaluate quantitatively the gene expression of enzymes SAP-2, SAP-5 and SAP-9 of the biofilm formed in internal surface of rat`s upper acrylic plates, and to analysis microscopically, the fungal invasion in palatal epithelial tissue. 49 Wistar rats were selected, 90 days old, weighing on average 300g. They were divided into three groups: Control Group, Plate/Candida and Plate, follow by 2, 4 and 6 days of the use of the plates. The results demonstrated, in four days of use of the acrylic plate, clinical signs of inflammation in the hard palate; microscopically epithelial hyperplasia, hyperkeratinization, fungal invasion in the superficial layers of the epithelial lining, Munro`s microabscess and papillary hyperplasia; and higher percentage of neutrophils in the Plate/Candida Group, compared to Control and Plate Groups. In the period of 4 days, the relative gene expression of the SAPs-2, -5 and -9 in biofilm also showed to be higher in Plate/Candida Group, compared with the periods of 2 and 6 days. Thus, the fungal invasion in the epithelial lining of the hard palate may be correlated with high mRNA expressionn of SAPs -2, -5 e -9.