Thesis (S.M.)--University of Chicago, Department of Microbiology. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (S.M.)--University of Chicago, Department of Microbiology. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Orientador: Eunice Teresinha Giampaolo / Banca: Gisele Faria / Banca: Elaine Maria Sgavioli Massucato / Banca: Janaina Habib Jorge / Banca: Maria Aparecida de Andrade Moreira Machado / Banca: Dalva Cruz Laganá / Resumo: A Candida albicans, presente nos biofilmes orais, é a principal responsável pela estomatite protética. Normalmente essa infecção é assintomática, porém, quando os sinais e sintomas estão presentes podem causar sangramento das mucosas, edema local, ardor ou outras sensações dolorosas. Os medicamentos tópicos, como a nistatina e o miconazol, têm sido utilizados, entretanto, o efeito diluente da saliva, movimentação da língua e da musculatura bucal reduzem a dose desses agentes a concentrações subterapêutica, além do alto índice de recorrência da infecção. Assim, é importante a procura por novos antifúngicos, que sejam eficientes e não tóxicos. A cavidade bucal é constantemente banhada de saliva, um fluido biológico com potente atividade antifúngica e bacteriana. Com o recente progresso na análise do proteoma salivar, o número de proteínas salivares identificado aumentou consideravelmente. A Histatina 5 gerada proteoliticamente a partir de Hst-3, durante a maturação dos grânulos nas glândulas salivares das parótidas tem sido extensivamente estudada devido a sua maior ação fungicida sua notável ação contra a C. albicans. Métodos baseados em medidas da atividade metabólica e viabilidade celular, como o ensaio de redução do sal XTT (hidróxido de 2,3-bis (2-metoxi-4-nitro-5-sulfofenil) -5 - [(fenilamino) carbonil] -2H-tetrazólio ) e contagem de unidades formadoras de colônia (UFC) tem sido utilizados para a avaliação de novos medicamentos capazes de inibir a formação do biofilme. Por serem feitos separadamente, esses ensaios demandam tempo, alto custo e variabilidade entre as amostras. Assim, este estudo teve como objetivo avaliar inicialmente, i) os efeitos do sal XTT e os demais compostos utilizados para realizar o ensaio de redução de XTT, sobre a viabilidade celular dos biofilmes de C. albicans e S. Mutans; ii) avaliar o potencial da histatina...(Resumo completo clicar acesso eletrônico) / Abstract: Candida albicans, present in oralbiofilms, is primarily responsible for denture stomatitis. Usually the infection is asymptomatic, but when signs and symptoms are present it can cause mucosal bleeding, swelling, burning or other painful sensations. Topical drugs such as miconazole and nystatin have been used, however, the diluent effect of saliva, the tongue and oral muscles movements reduce the dose of these agents to subtherapeutic concentrations, besides the high rate of recurrence of the infection. Thus, it is important to search for new antifungals, which are efficient and non-toxic. The oral cavity is constantly in presence of saliva, a biological fluid with potent antifungal and bacterial activity. With the recent progress in analysis of saliva proteome, the number of identified salivary proteins has increased considerably. The histatin-5 generated proteolytically from Hst-3, during maturation of the granules in the salivary glands of the parotid has been extensively studied due to their greater fungicidal action his remarkable action against C. albicans. Methods based on measurements of metabolic activity and cell viability as reduction assay salt XTT (2,3-bis hydroxide (2-methoxy-4-nitro-5-sulfophenyl) -5 - [(phenylamino) carbonyl] - 2H-tetrazolium) and counting of colony forming units (CFU) have been used for the exploration of new drug potential on inhibitory development of biofilm. The associations between the metabolic activities experiments with counting CFU are labor-intensive processes that need to be done separately. This study aimed to evaluate initially i) the effects of XTT salt and other compounds used to make the XTT reduction assay on cell viability of biofilms of C. albicans and S. mutans; ii) evaluate the potential of histatin 5 in protecting the human oral epithelium against C. albicans and iii) identify the heterotypic complexes formed between the histatin 5, a natural...(Complete abstrat electronic access below) / Doutor

Orientador: Cristiane Yumi Koga-Ito / Co-orientador: Marcos José Salvador / Banca: Janete Dias Almeida / Banca: Sônia Khouri / Banca: Fernanda Lourenção Brighenti / Banca: Antônio Carlos Victor Canettieri / Resumo: O objetivo deste estudo foi avaliar a efetividade frente a Candida albicans da fração enriquecida em fenólicos (FE) das folhas de Buchenavia tomentosa além das substâncias ácido gálico (AG), corilagina, kaempferol e vitexina, substâncias fenólicas que foram previamente detectadas no extrato aquoso de B. tomentosa livres ou encapsuladas em ciclodextrinas. Para tal, foi realizado teste de microdiluição com cepas padrão e isolados clínicos e análise química da FE por espectrometria de massa (ESI-MS). Detectou-se que o extrato acetônico foi a FE e AG foi a substância fenólica mais eficiente contra C. albicans. O efeito de FE e AG contra os fatores de virulência de C. albicans foram analisados. FE e AG foram encapsulados em 2-hidroxipropil-beta-ciclodextrina e tiveram sua análise química realizada. As CIMs e CFMs dos encapsulados foram determinadas, porém apenas o AG encapsulado teve sua ação antibiofilme e in vivo verificadas. A citotoxicidade de AG e FE livres e encapsulados foi determinada. As CIMs variaram de 5,0 e 0,625 mg/ml para o ácido gálico e 2,5 e 0,019 mg/ml para FE. AG e as outras moléculas foram encontradas na FE. Não foram encontrados CFMs. Os fenólicos estudados também foram encontrados em FE por ESI/MS. Tanto FE quanto AG tiveram efeito direto nos fatores de virulência de C. albicans, exceto sobre a secreção de exoenzimas. Não houve diferença na CIM entre as substâncias livres e encapsuladas. AG encapsulado teve melhor ação anti-biofilme do sua forma livre. Foi verificada melhora clínica de lesões eritematosas no palato de ratos, porém não foi possível. A citotoxicidade das substâncias livres ou encapsuladas variou de moderava a leve para FE e foi moderada para AG. Após as análises, observou-se o efeito anti-C. albicans de FE e AG. AG encapsulado apresentou promissor efeito anti-biofilme e aparente melhora clínica nas lesões sugestivas de candidose eritematosa na mucosa palatar dos ratos / Abstract: The aim of this study was to evaluate the effectiveness against Candida albicans of fraction enriched in phenolic (FE) of Buchenavia leaves tomentosa beyond substances gallic acid (GA), corilagin, kaempferol and vitexin, phenolic substances previously detected in the aqueous extract of B. tomentosa, free or encapsulated in cyclodextrins. Microdilution test with standard strains and clinical isolates besides the chemical analysis of FE by mass spectrometry (ESI-MS) were carried out. The acetone extract was the FE and AG was the most efficient phenolic substance against C. albicans. The effect of FE and AG against the virulence factors of C. albicans was also analyzed. FE and AG were encapsulated into 2- hydroxypropyl-beta-cyclodextrin (HP-β-CD) and had their chemical analyses made. MICs and MFCs of encapsulated have been determined. Solely the GA encapsulated had its anti-biofilm and in vivo action verified. Cytotoxicity of free and encapsulated GA and FE were determined. The MIC ranged from 5,0 to 0,625 mg/ml for GA and 2,5 and 0,019 mg/ml for FE. MFCs values were not found. All phenolics molecules were found in FE by ESI/MS. AG and FE had a direct effect on virulence factors of C. albicans, except on the secretion of exoenzymes. There was no difference in the CIM between free and encapsulated substances. The anti-biofilm effect was better in GA encapsuladed than its free form. A clinical improvement of sugestives erythematous lesions on the palate of rats was observed, although the hyphaes were not found in the palatar mucosa. The cytotoxicity for all substances was moderated. After the analysis, we observed the anti-C. albicans effect of GA and FE. AG encapsulated showed promising anti-biofilm effect and apparent clinical improvement in lesions suggestive of erythematous candidiasis in palatar mucosa of rats / Doutor

No description available.

published_or_final_version / Dentistry / Master / Master of Dental Surgery

published_or_final_version / abstract / Dentistry / Doctoral / Doctor of Philosophy

The main objective of this thesis was to study the effects of glucose on the regulation central carbon metabolic functions in the human fungal pathogen Candida albicans (C. albicans). The virulence of C. albicans is dependent upon fitness attributes as well as virulence factors. These attributes include robust stress responses and metabolic flexibility (Brown, 2005). The assimilation of carbon sources is fundamentally important for growth in all organisms and essential for the establishment of infections by pathogens, such as C. albicans in their human host. The C. albicans PCK1 and ICL1 genes, which encode the gluconeogenic and glyoxylate cycle enzymes phosphoenolpyruvate carboxykinase and isocitrate lyase are required for growth on non-fermentable carbon sources such as lactate and oleic acid. This thesis examines the impact of glucose upon the assimilation of secondary carbon sources such as lactate and oleic acid by C. albicans. The addition of 2% glucose repressed the CaPCK1 and CaICL1 genes. However, the enzymes CaIcl1 and CaPck1 were not destabilised by glucose and they retained at high levels following glucose addition. As a result, C. albicans cells continued to assimilate lactate and oleic acid in the presence of glucose. In contrast, the ScPck1 and ScIcl1 proteins were degraded rapidly in S. cerevisiae, and lactate and oleic acid assimilation was repressed in response to glucose. Therefore, while C. albicans and S. cerevisiae display similar responses to glucose at the transcriptional level, their responses at post-transcriptional and metabolic level diverge significantly. As a result, C. albicans can assimilate both glucose and alternative carbon sources at the same time. Next, the molecular apparatus that triggers the destabilisation of target proteins in response to glucose in C. albicans was studied. The expression of C. albicans the ICLI ORF in S. cerevisiae suggested that CaIcl1 has lost the molecular signal that triggers destabilisation in response to glucose, as CaIcl1 was not degraded in response to glucose in S. cerevisiae. However, when ScIcl1 was expressed in C. albicans ScIcl1 was rapidly degraded in response to glucose indicating that C. albicans has retained the molecular apparatus for glucose-accelerated degradation of target proteins. ScIcl1 degradation was slowed in C. albicans ubi4/ubi4 mutants. Furthermore, the addition of a putative of S. cerevisiae ubiquitination site carboxy terminus of CaIcl1 led to glucose-accelerated degradation of this protein in C. albicans cells. Therefore, glucose triggers accelerated degradation of target proteins in C. albicans via a ubiquitin-dependent process.

Candida albicans, a successful human pathogen, displays the phenomenon of glucoseenhanced oxidative stress resistance (Rodaki et al., 2009), which is not observed in other yeast species tested. The molecular bases of the phenomenon are not clear. It was suggested that glucose signalling might play a major role. Therefore, the impact of specific C. albicans mutations was tested to determine which of three known major glucose signalling pathways are required for glucose-enhanced oxidative stress resistance. Two major glucose signalling pathways were found to contribute to the phenomenon (the glucose repression pathway and the cAMP pathway), and a third pathway (the SRR pathway) is not essential for this response. The next step was to identify targets of these pathways that might contribute to the phenotype. First, it was tested whether known oxidative stress systems contribute to the GEXSR. The selected targets represented almost all main systems involved in redox control and ROS detoxification (catalase, superoxide dismutases, thiredoxins, and glutathione peroxidases) which seemed to contribute not significantly to the GEXSR. The exceptions to this were glutathione reductase (Glr1) and glutaredoxin (Grx2/Ttr1), inactivation of which affected manifestation of the phenomenon. This reinforced the view that the GSH/GSSG balance plays a key role in the GEXSR. Second, comparative analyses of transcriptomic profiles of C. albicans glucose- and lactategrown cells in response to oxidative stress and glucose treatment correspondingly revealed a small set of commonly up-regulated genes: UCF1, RNR22, MOH1, orf19.3302, and HSP21/orf19.822 (Enjalbert et al., 2006; Rodaki et al., 2009). Each potential GEXSR-specific gene appeared to be regulated in a distinct manner by the major glucose signalling pathways. The ectopic expression of potential GEXSR targets did not provide any experimental evidence to support their roles in this response. That might be related to inefficient expression from ACT1 promoter under the experimental conditions tested, and also caused by other effects.

Disruption of <i>N-</i>mannosylation and <i>O</i>-mannosylation on the <i>C. albicans</i> outer cell wall increased the rate by which <i>C. albicans</i> is ingested by macrophages. Conversely, disruption of phosphomannosylation reduced the rate of <i>C. albicans</i> is phagocytosis. Alterations to the outer cell wall and genetic or chemical inhibition of hyphal morphogenesis in <i>C. albicans</i> resulted in significantly abrogated macrophage killing <i>in vitro</i>. Disruption of <i>C. albicans </i>ability to tolerate oxidative stresses also perturbed its ability to escape from and kill macrophages. The engagement of specific receptors on the macrophage surface is an essential component of <i>C. albicans</i> recognition and clearance. In the presence of serum, blocking pattern recognition receptors associated with specific fungal cell wall epitopes (Mannose Receptor, Dectin 1 and CD16/32) resulted in an initial decrease in phagocytosis and decreased macrophage killing. Blocking macrophage pattern recognition receptors using soluble components of the<i> C. albicans</i> cell wall resulted in decreased phagocytosis under serum free conditions of <i>O-</i>linked mannans only, and reduced macrophage killing for macrophages pre-exposed to <i>N-</i>mannan and laminarin. The presence of serum increased the rate of uptake for macrophages pre-exposed to <i>N-</i>mannan and laminarin, and had no affect upon macrophage killing. The interaction of <i>C. albicans</i> cell wall epitopes with macrophage pattern recognition receptors, coupled with <i>C. albicans</i> ability to respond to stresses encountered after ingestion are critical determinants of the macrophage’s ability to ingest and process <i>C. albicans.</i>