Orientador: Marta Cristina Teixeira Duarte / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-26T04:57:52Z (GMT). No. of bitstreams: 1 Matsumura_LauraYumeRodrigues_M.pdf: 1312693 bytes, checksum: 7afc440e71f8e63ffdb4e0f57d78115f (MD5) Previous issue date: 2014 / Resumo: As leveduras causam deterioração de uma grande variedade de produtos alimentícios e nas indústrias de bebidas, além de serem resistentes a muito conservantes químicos. Os óleos essenciais e extratos de plantas têm surgido como alternativas seguras para substituir conservantes sintéticos. Contudo, os mecanismos de ação de óleos essenciais sobre micro-organismos são complexos e não estão completamente elucidados. Neste trabalho, foi investigada a ação de 14 óleos essenciais e 2 extratos diclorometânicos provenientes de plantas medicinais e aromáticas pertencentes à CPMA (Coleção de Plantas Medicinais e Aromáticas) do CPQBA/UNICAMP sobre leveduras do gênero Candida e sobre Pichia guilliermondii de importância em alimentos. Foi determinada a concentração mínima inibitória (MIC) e os efeitos sobre a lise da membrana celular, sobre os carboidratos de reserva trealose e glicogênio, sobre a depleção de ATP e sobre a biossíntese de ergosterol. Os resultados demonstraram que o óleo essencial de Cinnamomum burmanni foi o que apresentou melhor potencial para controle das leveduras, sendo o cinamaldeído e o acetato de cinamila os compostos majoritários presentes neste óleo. A investigação dos possíveis mecanismos de ação do óleo essencial de C. burmanni sobre Candida albicans ATCC 10231 demonstrou que este afetou a viabilidade da levedura a partir da concentração de 0,5 mg/mL (5MIC), e ocasionou a lise da membrana celular, havendo liberação de proteínas e lipídios para o meio extracelular, além de depleção de ATP. No caso dos carboidratos de reserva, os resultados demonstraram que o óleo essencial de C. burmanni ocasionou acúmulo de trealose, possivelmente pelo estresse ocasionado às células. Nenhum efeito foi observado sobre a reserva de glicogênio e sobre a inibição da síntese de ergosterol. Os resultados indicam a ação inibitória do óleo essencial de C. burmanni e mostram que este apresenta potencial para controle de leveduras de importância em alimentos / Abstract: Yeasts cause deterioration of a wide variety of food and drink industries, besides being very resistant to chemical preservatives. Essential oils and plant extracts have emerged as safe alternatives to synthetic preservatives. However, the mechanisms of action of essential oils on microorganisms are complex and not fully elucidated. In this work, was investigated the action of 14 essential oils and 2 dichloromethane extracts from medicinal and aromatic plants belonging to the Collection of Medicinal and Aromatic Plants - CPMA at CPQBA/ UNICAMP on Candida species and Pichia guilliermondii of importance in foods. The minimum inhibitory concentration (MIC) was determined and the effects of the essential oil on the cell membrane lysis, the carbohydrate reserves trehalose and glycogen, depletion of ATP and ergosterol biosynthesis were evaluated. The results showed that the essential oil from Cinnamomum burmanni presented the best potential to control the Candida spp. being the cinnamaldehyde and cinnamyl acetate the major compounds present in this oil. The investigation of the possible mechanisms of action of the C. burmanni essential oil on Candida albicans ATCC 10231 showed that the oil affected the viability of the yeast from 0.5 mg/mL (5MIC), and caused lysis of the cell membrane, with release of proteins and lipids into the extracellular environment, as well as ATP depletion. In the case of carbohydrate reserves, the results showed that the essential oil of C. burmanni caused accumulation of trehalose, possibly due to the cellular stress. No effect was observed on the synthesis of glycogen and ergosterol. The results indicate the inhibitory action of the essential oil from C. burmanni and show its potential to control yeasts of importance in foods / Mestrado / Ciência de Alimentos / Mestra em Ciência de Alimentos

Orientador: Altair Antoninha Del Bel Cury / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-19T16:58:30Z (GMT). No. of bitstreams: 1 Custodio_William_D.pdf: 6418945 bytes, checksum: f47ba0f315d5085af1b6afa2e2734527 (MD5) Previous issue date: 2012 / Resumo: O desenvolvimento de biofilme de Candida albicans pode ser mediado pela expressão diferencial de sítios de ligação protéicos na película adquirida formada sobre as superfícies das próteses dentais. Assim, objetivo geral deste estudo foi verificar a influência das proteínas de origem salivar e plasmática na formação dos biofilmes de C. albicans. No primeiro capítulo foi revisado o estado da arte de metodologias aplicadas para análise de proteínas. A partir do conhecimento das metodologias, a pesquisa foi realizada com objetivo de caracterizar os perfis protéicos de películas adsorvidas (ADP) à superfície de espécimes de poli(metilmetacrilato), na presença de saliva ou saliva acrescida de plasma sanguíneo. A composição da ADP foi analisada utilizando a técnica de espectrometria de massas, cujo resultado demonstrou diferenças significativas nos proteomas obtidos entre os grupos. Ainda, foi verificada a influência destas películas na energia livre de superfície (SFE) e expressão de fatores de virulência de biofilmes de C. albicans. Os achados demonstraram que a ligação de proteínas plasmáticas determinou aumento de ambas variáveis. A partir desses dados estudou-se o efeito individual de algumas das proteínas identificadas na ADP no desenvolvimento de biofilmes de C. albicans. Com esse objetivo, biofilmes de C. albicans foram desenvolvidos (90 min, 24, 48 e 72 h) sobre películas mono protéicas de albumina, mucina I e II, lactoferrina, fibrinogênio, C3b, IgA e histatina 5. Em cada um dos tempos foi avaliada a atividade metabólica da célula fúngica (teste de XTT), a organização estrutural do biofilme (microscopia confocal por varredura à laser) e a expressão de fatores de virulência por meio dos testes de produção enzimática. Baseado nas evidências geradas por estes trabalhos, podese concluir que proteínas salivares e plasmáticas adsorvidas como integumentos protéicos mantêm sua função biológica e, assim, exercem uma influência modulatória no desenvolvimento de biofilmes de C. albicans / Abstract: The development of Candida albicans biofilms may be mediated by a differential expression of proteic ligand sites in the acquired pellicle formed onto the oral prosthesis surfaces. Therefore, the main objective of this study was to evaluate the influence of salivary and plasmatic-derived proteins on the development of C. albicans biofilms. In the first chapter the state-of-the-art was reviewed for methodologies applied to the analysis of proteins. From the knowledge of the methodologies, the research was realized aiming to characterize the protein profile of acquired pellicles (ADP) on poly(methylmetacrilate) surfaces' specimens in the presence of saliva or saliva supplemented with blood plasma. ADP composition was analyzed by mass spectrometry techniques, which results demonstrated a significant difference in the obtained proteome between the groups. Moreover, the influence of these pellicles was evaluated for the surface free energy (SFE) and expression of virulence factors by C. albicans biofilms. The findings demonstrated that the binding of plasmatic proteins in the ADP determined increases in both experimental variables. From these data, the individual effect of some identified ADP proteins was evaluated on C. albicans biofilm development. With this objective, C. albicans biofilms were developed (90 min, 24, 48 and 72 h) over mono-protein pellicles of albumin; mucin I and II; lactoferrin; fibrinogen; C3b; IgA and histatin 5. For each time point, biofilms were analyzed for metabolic activity (XTT assay), structural biofilm conformation (Confocal scanning laser microscopy) and, expression of virulence factors by means of enzymatic activity assays. Based on the generated evidences of these studies, we can conclude that adsorbed salivary and plasmatic proteins as proteic integuments keep their biological function, and thus exert a modulatory influence on C. albicans biofilm development / Doutorado / Protese Dental / Doutor em Clínica Odontológica

Orientador: Altair Antoninha Del Bel Cury / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-21T19:13:49Z (GMT). No. of bitstreams: 1 Lucena_SilviaCarneirode_D.pdf: 2411404 bytes, checksum: 60623a707d801134f36a5179acd0d512 (MD5) Previous issue date: 2013 / Resumo: A adequada higienização das próteses dentais removíveis é fundamental para prevenir o acúmulo de biofilme, que funciona como reservatório de microorganismos que podem levar a infecções locais e sistêmicas. Apesar de a escovação constituir um método bem estabelecido para tal fim, limitações visual e motora de alguns pacientes reduzem sua eficácia. Assim, o uso de limpadores químicos (LQ) tem sido indicado como método complementar para remoção do biofilme. Frente ao exposto, os objetivos deste trabalho foram (i) avaliar in vitro o efeito da imersão diária em LQ sobre biofilmes multiespécies desenvolvidos em resina para base de próteses removíveis e (ii) avaliar in vivo o efeito do LQ no controle do biofilme de próteses parciais removíveis (PPR). Para o estudo in vitro, foram confeccionados discos de resina acrílica sobre os quais foram desenvolvidos biofilmes multiespécies (Candida albicans, Veillonella dispar, Streptococcus mutans, Streptococcus oralis, Fusubacterium nucleatum e Actinomyces naeslundii). Após 64,5 horas de desenvolvimento do biofilme, os espécimes foram aleatoriamente distribuídos em grupos controle e experimental. Os espécimes do grupo experimental foram submetidos a imersões diárias de 3 minutos na solução de LQ (Polident® 3 minute) por sete dias consecutivos; o biofilme do grupo controle foi desenvolvido durante o mesmo período sem tratamento. O biofilme de ambos os grupos foi coletado após 1, 4 e 7 dias experimentais (n=16) e analisados quanto ao número de células viáveis e concentração de polissacarídeos. Adicionalmente, a topografia superficial do biofilme e a morfologia celular foram analisadas através de microscopia eletrônica de varredura e confocal a laser. Para a avaliação in vivo, foram selecionados 25 voluntários usuários de PPR. Estes foram instruídos a complementar a higienização das próteses imergindo-as na solução do LQ (Polident® 3 minute) diariamente, por 3 minutos, durante 15 dias. O biofilme presente nas próteses foi coletado imediatamente antes e após o período experimental e o número de micro-organismos totais, estreptococos totais e Candida spp. foi quantificado. No estudo in vitro, foi observado que a imersão diária no LQ reduziu significativamente o número de micro-organismos totais quando comparado ao grupo controle nos três períodos avaliados (Análise de variância a dois fatores, p<0,05). Esta redução também foi observada para as bactérias individualmente e F. nucleatum e V. dispar não foram detectadas nos biofilmes expostos ao LQ. Observou-se um aumento gradativo na contagem de células de C. albicans no grupo experimental, comportamento inverso ao grupo controle no qual houve redução do número de células fúngicas viáveis. A concentração de polissacarídeos extracelulares no biofilme foi maior no grupo experimental após sete dias de limpeza quando comparado ao controle (Análise de variância a dois fatores, p<0,05). As imagens de microscopia revelaram elevada quantidade de hifas de C. albicans no grupo experimental. No estudo in vivo, observou-se uma redução significativa do número de micro-organismos totais e estreptococos totais após o período experimental (Teste-t pareado, p<0,05). Entretanto, não houve diferença para as contagens de Candida spp.. Conclui-se que a exposição diária ao limpador químico reduziu significativamente os micro-organismos totais viáveis em biofilmes multiespécies mas não apresentou eficácia sobre Candida / Abstract: The maintenance of adequate denture hygiene is of paramount importance to prevent biofilm accumulation which acts as a reservoir of microorganisms that can cause local and systemic infections. Although brushing is a well established method for denture cleaning, visual and manual limitations of some patients can reduce its efficacy. Therefore, the use of denture cleansers (DC) has been indicated as a complementary method for denture hygiene. Considering this, the aims of this study were to (i) evaluate, in an in vitro model, the effect of daily immersion in DC on a multispecies biofilm develop on denture-base acrylic resin and (ii) assess the effect of DC on controlling biofilm from removable partial dentures (RPD) surfaces. For the in vitro study, specimens were fabricated using acrylic resin on which it was develop a multispecies biofilm (Candida albicans, Veillonella dispar, Streptococcus mutans, Streptococcus oralis, Fusubacterium nucleatum e Actinomyces naeslundii). After 64.5 hours of biofilm growth, the specimens were randomized into control and experimental groups. The specimens of experimental group were submitted to 3-minutes immersions on DC solution daily for seven consecutive days; the biofilms of control group were developed for the same period without any treatment. The biofilms from both groups were collected after 1, 4 and 7 experimental days (n=16) and analyzed for viable cells and polysaccharides concentration. Additionally, the biofilms topography and cellular morphology were analysed by scanning electron microscopy ) and confocal microscopy. For in vivo evaluation of DC, 25 volunteers that used RPD were selected. They were instructed to complement their dentures hygiene by immersing them on DC solution, for 3 minutes, during 15 days. The biofilm present on RPD was collected immediately before and after the experimental period and the number of total microorganisms, total streptococci and Candida spp. were quantified. In the in vitro study, it was observed that daily immersion on DC significantly reduced the total microorganisms in comparison to control group, in all periods evaluated. (Two-way Anova, p<0.05). This reduction was also observed for individual bacteria and F. nucleatum and V. dispar were not detected on biofilms exposed to DC. It was observed a gradual increase of C. albicans count in biofilms of experimental group while, in control group, Candida counts decreased and it was not observed viable cells after seven experimental days. Additionally, extracellular polysaccharides concentration was significantly higher in experimental group after 7 days of treatment (Two way Anova, p<0.05). The microscopy images revealed a remarkable presence of hyphae in experimental group. In clinical study, it was observed a reduction on total microorganisms and total streptococci. However, no difference was observed in Candida spp. counts (paired t-test, p<0.05). It can be concluded that daily exposure to DC reduced total microorganisms on multispecies biofilms but did no demonstrated efficacy on Candida / Doutorado / Protese Dental / Doutora em Clínica Odontológica

Os leucotrienos aumentam a fagocitose e a atividade microbicida contra uma série de patógenos. Em macrófagos alveolares, LTB4 e LTD4 aumentam a fagocitose via Fc&#947;R de modo dependente de PKC. Entretanto, o papel das isoformas específicas da PKC, das MAPK, e da PI3K neste processo, ainda não é conhecido. Além disso, pouco se sabe sobre a importância dos leucotrienos na fagocitose via outros receptores. Os objetivos deste trabalho são: a) ampliar o conhecimento sobre as vias de sinalização ativadas pelos leucotrienos durante a fagocitose de hemácias opsonizadas por IgG; b) avaliar o efeito dos leucotrienos na fagocitose de Candida albicans, por macrófagos alveolares e as vias de sinalização intracelular envolvidas. Observou-se que os leucotrienos endogenamente produzidos ou adicionados aos macrófagos alveolares, aumentam a fagocitose via Fc&#947;R e para isso utilizam distintas vias de sinalização intracelular. A ação do LTB4 envolveu predominantemente a via ERK1/2 e PKC&#945;. E com menor intensidade da PKC&#948;. A ação do LTD4 envolveu exclusivamente a via p38 e PKC&#948;. Ambos leucotrienos utilizaram a PI3K para aumentar a fagocitose via Fc&#947;R. Na fagocitose de C.albicans, os macrófagos alveolares produziram LTB4. Tanto o LTB4 quanto o LTD4 endógenos contribuíram para a fagocitose da C.albicans que ocorre predominantemente via receptor manose. A fagocitose via receptor manose foi potencializada pela adição tahto do LTB4 quanto do LTD4 c&#947; de modo dependente da ativação da PKC&#948; e da PI3K. As vias da PKC&#945;., ERK1/2 e p38 não estão envolvidas na potencialização da fagocitose de C.albicans por leucotrienos. O esclarecimento das moléculas sinalizadoras envolvidas na interação de macrófagos alveolares com patógenos poderá contribuir para o desenvolvimento de alvos específicos e refinados no controle e tratamento de infecções pulmonares. Analisados em conjunto, os resultados mostram que os leucotrienos são importantes moduladores das funções dos macrófagos alveolares, por aumentar a fagocitose tanto através dos receptores para Fc&#947; como do receptor manose. Além disso, os LTB4 e LTD4 ativam programas de sinalização distintos para potencializar a fagocitose via receptor Fc&#947;, enquanto na fagocitose via receptor manose os LTB4 e LTD4 usam as mesmas vias. / Leukotrienes enhance phagocytosis and microbicidal activity against several pathogens. In alveolar macrophages, LTB4 and cysteinyl LT both enhance Fc&#947; receptor - mediated phagocytosis. While both LTB4 and LTD4 enhances phagocytosis dependent on PKC activity, only LTB4 requires the protein tyrosine kinase syk to do so. However, the role of specific PKC isoforms, MAPKs (p38 and ERK1/2), and PI3K in mediating LT-enhanced Fc&#947;R-mediated phagocytosis is unknown. In addition, the importance of LT in the phagocytosis via other receptors is also understudied. In the present work we sought to determine the importance of the above kinases during IgG-opsonized phagocytosis and if both classes of LT enhance the ingestion of C. albicans; the receptors and the molecular mechanisms involved. Studies with isoform-selective inhibitors indicated that LTB4 effects were dependent on both PKC&#945. And PKC&#948;, while LTD4 effects were exclusively due to PKC&#948; activation. Although both exogenous LTB4 and LTD4 enhanced p38 and ERK1/2 activation, LTB4 required only ERK1/2, while LTD4 required only p38 activation. Activation by both LT was dependent on PI3K activation. It was found that exogenously added LTB4 and LTD4 both enhanced PKC& and PKC&#945;. Phosphorylation during Fc&#947;R engagement. Regarding the studies concerning the importance of LT in the ingestion of C.albicans, both endogenous and exogenous LTB4 and LTD4 enhanced C.albicans phagocytosis. LT effects were dependent exclusively on PKC&#948; and P13K. We also demonstrated that the LT effect on C.albicans was due to the mannose receptor activation. Taken together, leukotrienes are potente immunomodulators of the phagocyte function, by enhancing phagocytosis both via Fc&#947;R and mannose receptor. Moreover, LT receptors activate different signaling programs to enhance Fc&#947;R-mediated phagocytosis, while the signaling elicited to enhance C.albicans ingestion is similar for both classes of LTs. Thus the nature of the signaling elicited by the phagocytic receptors dictates the signaling induced by LT.

Candida albicans est un champignon polymorphe, membre de la flore normale humaine, où il réside comme un organisme commensal tout au long de la vie. Cependant, dans certaines circonstances il peut causer des mycoses qui vont des infections superficielles de la peau à des infections systémiques mortelles. Très peu de choses sont connues sur les différences au sein des mécanismes fondamentaux de la croissance cellulaire, comme la synthèse des protéines par exemple, chez C. albicans par rapport aux organismes modèles tel Saccharomyces cerevisiae. À titre d'exemple, il a été démontré que son code génétique ne répond pas au code génétique universel. En effet, le codon CUG, codant normalement une leucine, code une sérine dans ces espèces, en particulier dans les protéines peu exprimées à la surface de la cellule, influençant potentiellement l'interaction de l'organisme avec l'être humain. La compréhension de la façon dont le mécanisme de traduction a lieu dans C. albicans est donc d'une importance cruciale. Le ribosome est la machinerie cellulaire responsable de la biosynthèse des protéines. Trouvé dans chaque organisme vivant, sa fonction est conservée, bien que sa structure puisse varier. Ces variations sont les particularités que nous cherchons à mettre en évidences, en effet elles sont les premières pistes de réponses aux questions concernant le mécanisme de traduction, potentiellement différentes de ce que l’on connait, de plus ces différences constitueront une base solide dans le développement de nouvelles molécules antifongiques. Le premier aspect de cette thèse tente de révéler l'intimité de la structure du ribosome 80S de C. albicans à haute résolution par modélisation in silico et confirmée par une approche de cristallographie aux rayons X. Ce modèle ouvrira la voie à d'autres études structurales de complexes fonctionnels, afin de déterminer la mécanique de la traduction des protéines dans cet organisme. Le deuxième aspect concerne l'inhibition du ribosome de C. albicans. Afin d'identifier des cibles intéressantes, des tests de concentration minimale inhibitrice ont été établies afin de confronter nos hypothèses structurales et de permettre d’évaluer l’efficacité de molécules inhibitrices et de guider le développement de nouveaux médicaments ciblant spécifiquement le ribosome de C. albicans. / Candida albicans is a polymorphic fungus, member of the normal human microbiome, where it resides as a lifelong, harmless commensal organism. Under certain circumstances, however, it can cause infections that range from superficial infections of the skin to life-threatening systemic infections. Very little is known about differences in fundamental mechanisms of cell growth, like protein synthesis for instance, in C. albicans compared to model systems, like Saccharomyces cerevisiae. As an example, it has been shown that its genetic code is not exactly the same as the universal genetic code. Indeed, the CUG codon, which normally specifies leucine, specifies serine in these species, especially in proteins expressed at a low level at the cell surface, potentially influencing the interaction of the organism with the human host. The understanding of how the translation mechanism takes place in C. albicans is therefore of pivotal importance. The ribosome is the cellular machinery responsible for protein biosynthesis. Found in every living organism, its function is conserved, although its structure can vary. These variations are especially what we would like to highlight, since they are indeed the first elements to respond to questions concerning the translation mechanism, potentially different from what is known, and these differences will be a solid foundation in the development of new antifungal molecules. The first aspect of this thesis attempts to reveal the intimacy of the C. albicans 80S ribosome structure at medium high resolution by in silico modelization and confirmed by an X-ray approach. This model will then pave the way for further structural studies of functional complexes, in order to unravel the protein translation mechanism in this organism. The second aspect concerns the inhibition of the C. albicans ribosome. In order to identify interesting druggable spots, MIC assessments have been set so as to justify our hypothesis and to allow for future screening tests and the design of new drugs specifically targeting the C. albicans ribosome.

La maladie de Crohn (MC) est une maladie inflammatoire chronique de l’intestin qui peut être expliquée par une dysbiose et une dérégulation de la réponse immunitaire. La mannose-binding lectin (MBL), récepteur lectinique et les Toll-like récepteurs jouent un rôle crucial dans la défense contre les pathogènes et dans le développement de la réponse inflammatoire. Ils peuvent reconnaitre de nombreux micro-organismes dont Candida albicans. Cette levure commensale est un immunogène des anticorps anti-Saccharomyces cerevisiae (ASCA), anticorps utilisés dans le diagnostic de la MC. Une association entre un déficit en MBL et des taux élevés d’ASCA a été montrée chez les patients atteints de MC. Ce déficit en MBL est fréquemment associé à un phénotype sévère de la maladie. Le récepteur TLR2, associé à TLR1 ou à TLR6, est également impliqué dans la reconnaissance de C. albicans et le maintien de la barrière intestinale. L’objectif de ce projet a été d’étudier le rôle de la MBL et des récepteurs TLR2/TLR1/TLR6 dans l’homéostasie intestinale et l’élimination de C. albicans du tube digestif. Pour cela, nous avons exploré, les effets de la MBL et des TLR sur la colonisation intestinale à C. albicans et l’inflammation intestinale. Par ailleurs, nous avons déterminé, chez l’homme, l’influence des polymorphismes du gène MBL2 sur la modulation de l’activité et des taux de MBL au cours de la MC.A l’aide d’un modèle murin, nous avons mis en évidence l’expression de MBL-A et de MBL-C par les cellules épithéliales intestinales et leur implication dans l’homéostasie intestinale. Un déficit en MBL favorise la colonisation par C. albicans et une dissémination de la levure, en présence d’une colite chimio-induite. Les récepteurs TLR1 et TLR2 participent également dans la défense contre la colonisation par C. albicans, sans pour autant que leurs déficits n’entrainent de dissémination de la levure. A l’inverse, le récepteur TLR6 favorise la colonisation par C. albicans. La MBL et les récepteurs TLR1/TLR2/TLR6 régulent aussi l’expression des cytokines pro-inflammatoires impliquées dans les réponses Th1 et Th17. Ces résultats expérimentaux ont incité à envisager la poursuite de notre travail en approfondissant l’étude clinique sur des variations quantitatives et qualitatives de la MBL chez les patients atteints de MC et leur lien éventuel avec la persistance de l’inflammation intestinale. L’étude sur la cohorte de patients MC a montré que le gène MBL2 était associé à un déficit quantitatif en MBL et un déficit qualitatif du complexe MBL-MASP chez les sujets sains et les patients. Ce polymorphisme est également associé à des taux élevés d’ASCA chez les patients MC (p<0.05) de la maladie et il est fréquemment associé aux formes sévères de la maladie. De plus, le variant rs2066844 du gène NOD2, variant associé à la MC, est significativement corrélé à une diminution de l’activité fonctionnelle du complexe MBL-MASP, sans diminution quantitative des taux de MBL.Ces données montrent, pour la première fois, la production intestinale de MBL, qui est modulée par la colonisation par C. albicans. Elles confirment le rôle de la MBL et des récepteurs TLR dans l’homéostasie intestinale et dans la défense contre C. albicans. De plus, l’étude clinique sur la cohorte de patients MC révèle un défaut d’activité fonctionnelle du complexe MBL-MASP chez les patients MC ayant des polymorphismes des gènes MBL2 et NOD2, pouvant mener à un phénotype sévère de la maladie. Ces études expérimentales et cliniques apportent des nouvelles données sur le lien entre les récepteurs de l’immunité innée et la maladie de Crohn ainsi que la colonisation/infection fongique et ouvrent des nouvelles perspectives sur le lien entre la persistance de la colonisation fongique et le déficit quantitatif/qualitatif de la MBL chez les patients MC. / Crohn’s disease (CD) is an inflammatory bowel disease characterized by a dysregulation of the inflammatory response caused by dysbiosis and immune system disorders. Mannose Binding Lectin (MBL) and Toll like receptors (TLR) are involved in recognition of microorganisms and inflammatory response. They can recognize different patterns on the surface of the pathogens including Candida albicans. This pathogenic yeast is an immunogen for anti-Saccharomyces cerevisiae antibody (ASCA), diagnostic marker of CD. An association between MBL-deficiency and ASCA is observed in CD, and this MBL deficiency is frequently associated with a severe clinical phenotype of CD. In addition to MBL, TLR2, which forms heterodimers with either TLR1 or TLR6, have a major role in the innate immune defense against C. albicans and promotes intestinal homeostasis. In this project, we studied the role of MBL, TLR1, TLR2, and TLR6 in intestinal homeostasis and elimination of C. albicans in the intestinal tract. In the first part this study; we assessed the effect of either MBL or TLR deficiencies on C. albicans colonization and intestinal inflammation in a murine model. In the second part of this study, we assessed the role of MBL polymorphisms in the modulation of MBL level and activity in CD patients.In murin model, we showed that MBL is locally produced by the epithelial cells in response to C. albicans sensing and to intestinal inflammation and this lectin is required for the intestinal homeostasis. MBL-deficient mice had a higher level of colonization than wild-type mice. DSS-induced colitis promoted a high C. albicans colonization and dissemination to the kidneys and lungs of MBL-deficient mice. MBL-deficient mice exhibited reduced expression of Il-1β and IL-6 and elevated expression of IL-17, IL-23, dectin-1 and TLR-4. In terms of mice deficient in TLRs, DSS treatment and C. albicans oral challenge induced greater weight loss, worse clinical signs of inflammation, higher histopathologic scores, and increased mortality rates in TLR1-/- and TLR2-/- mice when compared to TLR6-/- and wild-type mice. Cytokine expression (TNF, IL-1β, IL-10, and IL-17A) was significantly increased in TLR1-/- and TLR2-/- mice, while they were decreased in TLR6-/- mice. In addition to the experimental studies, we observed in the clinical cohort of CD patients that MBL2 variant rs5030737 (codon 52) was associated with a low level of MBL that leads to impaired MBL-MASP functional activities in both CD patients and healthy subjects. Furthermore, this variant was also associated with a higher level of ASCA in CD patients (p<0.05). Increased ASCA levels were found in CD patients with stricture formation and penetrating disease complications, 42% and 21% respectively. Besides, we observed that NOD2 variant rs2066844, associated with susceptibility to CD, is significantly correlated with the impairment of the functional activity of MBL-MASP complex.Overall, this study emphasizes the role of MBL and TLR in intestinal homeostasis and host defense against C. albicans and shows for the first time that MBL could be produced locally in the intestinal epithelial cells in response to C. albicans sensing. In terms of the clinical study, we observed that CD patients with a severe clinical phenotype have an impairment in MBL-MASP functional activity, and that this defect is associated with MBL2 and NOD2 polymorphisms These experimental studies contribute to understand the link between innate immunity receptors, Crohn’s disease and fungal colonization/infection. Finally, this study leads to new objectives which are to study the link between intestinal microbiota and MBL variation in Crohn’s disease patients.

Orientador: Rosilene Fernandes da Rocha / Banca: Maria Stella Amorim da Costa Zollner / Banca: Antonio Olavo Cardoso Jorge / Resumo: A Candidose bucal e um dos processos infecciosos micóticos mais comuns da cavidade oral e existe suscetibilidade aumentada para o mesmo durante o período neonatal principalmente devido à imaturidade dos mecanismos de defesa e a falta de uma microbiota bucal balanceada. Para verificar a presença de Candida spp. na cavidade bucal de lactentes foram examinados inicialmente cem bebês, nos quais foi feita coleta de material do dorso da língua com swab nas primeiras 24 horas de vida. Trinta e três recém-nascidos deste grupo foram acompanhados durante os primeiros quatro meses de vida sendo realizada mensalmente coleta de material da cavidade oral e avaliação das condições gerais de saúde, nutrição e higiene. A análise do material obtido nesse período mostrou positividade para Candida spp. em 64 (48,5%) das 132 amostras. A doença foi observada em 27,3% dos recém nascidos acompanhados. Candida albicans foi a espécie encontrada mais frequentemente (44,6%), e esteve presente em nove dos 11 casos de candidose bucal. Verificou-se com a ananmese que os possíveis fatores de risco para o grupo estudado foram o uso de chupeta e mamadeira, e, a introdução de outros tipos de alimento. Conclui-se que Candida albicans e a espécie prevalente nesta faixa etária / Abstract: Abstract: Oral candidosis is one of the most frequent mycotic infectious diseases of the oral cavity and there is increased susceptibility to this infection during the neonatal period due to immaturity of the defense mechanisms and the lack of a balanced buccal microbiota. To verify the presence of Candida spp. in the oral cavity of infants were examined initially a hundred babies from which were collected material of the lingual dorsum with swabs in the first 24 hours of life. Thirty-three newborn of this group were accompanied during the first 120 days of life and were submitted monthly to a material collection of the oral cavity and an evaluation of general conditions of health, nutrition and hygiene. The analysis of the material collected during this period showed positivity to Candida ssp. in 64 (48,5%) of the 132 samples. Disease was observed in 27,3% of those 33 newborns. Candida albicans was the most frequent species (44,6%) and it was present in nine of the 11 cases of oral candidosis. The anamnesis showed that the possible risk factors were pacifier use, bottle feeding and introduction of different food types. It was concluded that Candida albicans is the prevalent species in this age band / Mestre

A gomesina é um peptídeo antimicrobiano catiônico, purificado dos hemócitos da aranha caranguejeira Acanthoscurria gomesiana. Possui amplo espectro de atividade contra bactérias, fungos, protozoários e células tumorais. Candida albicans é uma levedura comensal que faz parte da microbiota humana. O tratamento desta micose geralmente é feito com fluconazol, contudo casos de resistência vêm sendo reportados, com isso vários peptídeos antimicrobianos vêm sendo estudados a fim de se tornarem tratamentos alternativos. Este trabalho teve como objetivo avaliar a eficácia do tratamento com a gomesina em um modelo de candidíase disseminada e vaginal. O tratamento de gomesina foi eficaz no controle do fungo. Verificou-se um efeito imunomodulatório, pois seu tratamento aumentou as concentrações de IL-6, TNF-<font face=\"symbol\">a e INF-<font face=\"symbol\">g dos rins dos animais com candidíase disseminada. A gomesina não apresentou nenhum efeito tóxico para os animais. Os dados apresentados neste estudo reforçam o potencial da gomesina para ser um agente. / The gomesin is a cationic antimicrobial peptide, purified from hemocytes of the spider Acanthoscurria gomesiana. It has a broad spectrum of activity against bacteria, fungi, protozoa and tumor cells. Candida albicans is commensal yeast that is part of the human microbiota. The treatment of this mycosis is usually done with fluconazole although cases of resistance have been reported. With the emergence of microorganisms resistance, several antimicrobial peptides have been studied in order to become alternative treatments. This study aimed to evaluate the effectiveness of treatment with gomesin in a model of disseminated and vaginal candidiasis. The treatment with gomesin showed to be effective in controlling the fungus. There was also found an immunomodulatory effect as its treatment increased concentrations of IL-6, TNF-<font face=\"symbol\">a and INF-<font face=\"symbol\">g in the kidneys of animals with disseminated candidiasis. The gomesin did not show any toxic effect to animals. The data presented in this study reinforce the potential of gomesin to be an antifungal agent.

É grande o interesse em substâncias que apresentem ação inibitória sobre leveduras do gênero Candida. Várias plantas utilizadas na medicina popular têm sido estudadas, com esta finalidade. O presente trabalho teve por finalidade avaliar a atividade inibitória in vitro de extratos de Arrabidaea chica (Verlot.), conhecida popularmente por crajirú em Rondônia (Brasil), sobre Candida albicans. Foi avaliada a concentração inibitória mínima (CIM) de extratos aquoso e hidroalcoólico, autoclavados e filtrados, utilizando a técnica de diluição em ágar, sobre vinte e cinco cepas de C. albicans. Os extratos brutos foram submetidos à Cromatografia de Camada Delgada (CCD). O extrato hidroalcoólico apresentou sete componentes e o aquoso autoclavado, dois. Tanto o extrato hidroalcoólico autoclavado quanto o filtrado inibiram todas as cepas de C. albicans na concentração de 20%. O extrato aquoso filtrado não apresentou efeito inibitório até a concentração de 50% e o extrato autoclavado, na concentração de 30%, inibiu 84% das cepas testadas. Os extratos, hidroalcoólico e aquoso, apresentaram atividade sobre C. albicans. O extrato hidroalcoólico apresentou desempenho superior, em menor concentração e maior número de componentes. / There is a great interest in substances that have inhibitory action on yeasts of the genus Candida. Several plants, used in popular medicine, have been studied for this purpose. This aim of this work was to evaluate in vitro the inhibitory activity of Arrabidaea chica (Verlot.) extracts, known in Rondônia (Brazil) as crajirú, against Candida albicans. The minimum inhibitory concentration (MIC) of aqueous and hydroalcoholic extracts, autoclaved and filtered was assessed, using the technique of agar dilution. Twenty-five strains of C. albicans were studied. The crude extracts were submitted to a Fine Chromatography Analysis (FCA). The hydroalcoholic extract presented seven compounds and the aqueous autoclaved extract two. Hydroalcoholic extracts inhibited all strains of C. albicans in the concentration of 20%. The aqueous filtered extract not showed inhibitory effect until 50%. The aqueous autoclaved extract, in the concentration of 30%, inhibited 84% of the strains. Hydroalcoholic and aqueous extracts showed activity against C. albicans. The hydroalcoholic extract presented superior performance in a lower concentration and had a greater number of compounds.

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.