Objectives: Antiadherent and antibacterial effects of certain plant extracts have been proven to be beneficial in preventive dentistry. In the present in situ/in vitro crossover study, the impact of plant extracts rich in polyphenols on the erosion-protective properties of the in situ pellicle was evaluated. Methods: Individual splints were prepared for 12 subjects for intraoral exposure of bovine enamel specimens. Following formation of a 1-min pellicle, watery plant extracts(leaves of the wild form of Ribes nigrum , the wild form of Origanum as well as a combination of both) were administered for 10 min in situ. Alternatively, a mouth rinse with fluorides (Elmex Kariesschutz) was performed for 1 min. After further oral exposure for 19/28 min, respectively, slabs were removed and incubated with HCl in vitro over 120 s (pH 2, 2.3, 3). The resulting calcium and phosphate release was quantified photometrically. Slabs with and without a 30-min in situ pellicle served as controls. The modification of pellicle ultrastructure was evaluated by transmission electron microscopy (TEM). Results: Plant extracts modulated the erosion-protective properties of the native in situ pellicle in all test groups in a pH-dependent manner. The combination of R. nigrum leaves and Origanum enhanced the protective properties of the pellicle at all pH values; the administration of this preparation was comparable, yet superior, to the effect of the fluoridated mouth rinse. TEM images indicated that rinsing with R. nigrum leaves /Origanum yielded a distinctly thicker and more electron-dense pellicle. Conclusion: The combination of certain plant extracts offers a novel approach to the complementary prevention of dental erosion.

Indiana University-Purdue University Indianapolis (IUPUI)School of dentistry / BACKGROUND: Fluoride and stannous ions have been reported to be relevant for dental erosion prevention. However, their interaction with the acquired dental pellicle (ADP), a clinically relevant erosion protective factor, is not well known and needs to be investigated. OBJECTIVES: To investigate the anti-erosive properties of fluoride-containing solutions and stannous solutions on enamel and dentin surfaces with a previously formed ADP. To characterize the protein profile of the ADP treated with the test solutions. METHODS: Phase I tested four solutions: SnCl2/NaF, NaF, SnCl2 and deionized water (DIW) (as negative control). Forty bovine enamel and dentin specimens 104 (4x4x2 mm3) were prepared and randomly distributed into 4 groups (n = 10). The specimens were incubated in clarified human saliva (CHS) for 24 h for pellicle formation and then they were subjected to a cycling procedure that included a 5-min erosive challenge (0.3-percent citric acid, pH 2.6); a 2-min treatment with the solution (between 1st, 3rd and 6th cycles); a 2-h immersion in CHS, and overnight immersion in CHS. Cycles were repeated 6x/day for 5 days. The outcome measure was surface loss (SL) using profilometry. Phase II: Thirty-two (32) bovine enamel specimens (882 mm3) (n = 8) were similarly prepared and incubated in saliva for 24 h and then treated with the solutions for 2 min followed by CHS immersion for 2 h. This cycle was repeated 3x for one day. The pellicles formed and treated with the test rinse solutions were collected, digested, and analyzed for specific protein content using liquid chromatography electrospray ionization tandem mass spectrometry (LCESI-MS/MS). RESULTS: Phase I: for enamel, SnCl2/NaF, SnCl2, NaF solutions provided 89 percent, 67 percent, and 42 percent SL reduction respectively compared with the control, while in dentin they provided 60 percent, 23 percent, and 36 percent, respectively, all significant at p < 0.05. Phase II: Seventy-two (72) common proteins were identified in all groups, 30 exclusive to DIW, 20 to SnCl2/NaF, 19 to NaF, and 13 to SnCl2. SnCl2/NaF increased the abundance of pellicle proteins than each one alone. CONCLUSION: SnCl2/NaF showed the best anti-erosive effect on both enamel and dentin. The findings suggest that the composition of acquired pellicle changes with different solutions, which may be related to their anti-erosive effect.

Shewanella oneidensis est une bactérie aquatique capable de chimiotactisme, c'est-à-dire d'orienter sa nage en réponse aux signaux qu'elle perçoit. Mon travail de thèse s'est focalisé sur l'étude du système chimiotactique de cette bactérie. Mon premier objectif a été d'identifier de nouveaux substrats induisant une réponse chimiotactique ainsi que les chimiorécepteurs les détectant. Une approche à haut débit utilisant une banque de substrats, couplée à une recherche de substrats plus spécifiques, a permis d'identifier de nouveaux signaux. Nous avons confirmé que S. oneidensis est attirée par les accepteurs alternatifs d'électrons ainsi que par certains métaux. Nous avons montré qu'elle est attirée par le malate et le chromate et repoussée par le nickel et le cobalt. Nous avons identifié deux MCPs potentiellement impliqués dans la détection du malate et un dans la détection du chromate.Mon second objectif a été de comprendre le rôle du système chimiotactique dans la formation d'un biofilm flottant : la pellicule. Nous avons montré que le développement de la pellicule est un processus en trois étapes déclenché par la détection de l'oxygène en condition statique, probablement par aérotactisme. Nous avons mis en évidence l'implication inattendue du chimiotactisme dans la formation de la pellicule. En effet des mutants délétés de gènes codant pour des éléments du système chimiotactique ne forment pas de pellicule normale. Le développement de la pellicule met en jeu d'autres signaux chimiotactiques, générés au sein de la pellicule, qui pourraient intervenir dans la localisation des cellules et l'homogénéisation de la pellicule au cours de sa maturation. / Shewanella oneidensis is an aquatic bacterium capable of chemotaxis, meaning that it can change its direction in response to detected signals. My thesis focused on the study of the chemotaxis system of this bacterium.The first objective of my thesis was to identify new substrates inducing a chemotactic response and the chemoreceptors involved in their detection. A high-throughput technique involving a library of solutes coupled with a search of more specific compounds allowed to identify new signals. We confirmed that S. oneidensis is attracted toward alternative electron acceptors and by several metals. We showed that S. oneidensis is attracted toward malate and also chromate. Finally, we identified two repellents, nickel and cobalt. After construction of deletion mutants, we identified two MCPs potentially involved in malate detection and one in chromate detection.The second objective of my thesis was to understand the role of the chemotaxis system in the formation of a floating biofilm: the pellicle. We first characterized the pellicle development through time, revealing that it is a three-step process. We then highlighted the unexpected role of the chemotaxis system in pellicle formation. Indeed mutants deleted of genes coding for chemotaxis elements are not able to form a pellicle or form an abnormal one. We showed that oxygen is the main signal triggering pellicle formation in static condition and that it is probably detected through aerotaxis. Pellicle development also involves other chemotactic signals produced in the pellicle which could be used in the localization of cells at the air-liquid interface but also in the homogenization of the pellicle.

La plupart du temps, les bactéries vivent au sein de biofilms : un tissu biologique accroché sur des surfaces (molles ou solides), qui est composé de bactéries et de matrice extracellulaire. Lors de cette thèse nous avons étudié les mécanismes qui contrôlent la formation d’un biofilm à l’interface eau-air par Bacillus subtilis (BS). D’abord, nous avons observé l’évolution phénotypique de BS au cours du développement de sa population en milieu liquide, dans des conditions de culture standard (pas de biofilm in fine). Nous avons constaté que la population exhibe différents types cellulaires (phénotypes) au cours du temps. Puis, afin d’observer les étapes de la formation d’un biofilm, nous avons créé une expérience qui permet de suivre l’évolution macroscopique de la concentration des bactéries et sa répartition spatiale au sein du milieu de culture. Nous constatons qu’une accumulation de bactéries se forme en dessous de l’interface eau-air avant même l’apparition d’un biofilms. Cette accumulation est concomitante avec de la convection dans le fluide (bioconvection). Le biofilm apparait lors de la phase de croissance de la population en bactérie pour une concentration moyenne dans le milieu de culture de l’ordre de 10¹³ bactéries/m³. Ensuite, nous avons formulé un modèle continu qui renseigne sur l’évolution de l’environnement des bactéries. Ce modèle reproduit la bioconvection observée dans les expériences et révèle son effet sur la concentration en dioxygène dissous dans la culture. Enfin, nous avons construit un modèle hybride continu-discret qui décrit la transition de bactéries déconnectées (nomades) vers des bactéries connectées formant un biofilm solide (sédentaires). Chaque bactérie est considérée comme une particule individuelle. Le modèle tient compte des forces de contact, ainsi que les forces élastiques qui peuvent s’établir entre les bactéries lorsqu’elles sont liées par de la matrice. Un nombre minimal d’aptitudes biologiques a été utilisé pour modéliser les bactéries : la division cellulaire qui leur permet de coloniser le milieu de culture, la motilité et l’aérotactisme qui explique leur migration vers la surface liquide, le quorum sensing (QS) et la différenciation cellulaire qui leur permet de passer du phénotype nomade (motile) au phénotype sédentaire (producteur de matrice). L’environnement en dioxygène des bactéries et les propriétés hydrodynamiques du milieu sont décrits par des champs continus. Le modèle reproduit toutes les étapes de la formation d'un biofilm observées dans nos expériences et confirme la nécessité de certaines aptitudes biologiques. Il montre que le seuil de QS joue un rôle majeur dans la morphologie du biofilm et sa cinétique de formation. En revanche, le taux de consommation de dioxygène par les bactéries ne semble pas avoir de rôle important. Enfin, nous avons établi que la bioconvection agit comme un retardateur de la formation du biofilm. / Most of the time, the bacteria live inside the biofilm: the biological tissue that is attached to the surface (soft or solid), is made of the bacteria and of extracellular matrix. According to this thesis we study the mechanics that control the formation of a biofilm at the interface water-air by Bacillus subtilis (BS). First, we absorbed the phenotype evolution of the BS during the development of its population in liquid medium, under the conditions of a standard culture (no of biofilm in fine). We noticed that the population exhibits different cellular types (phenotypes) during this time. Then, after absorbing the different stages of the development in the formation of a biofilm, we created an experience that allows us to follow the macroscopic evolution of the concentration of the bacteria and its special distribution in the middle of the culture. We found that an accumulation of the bacteria forms under the surface water-air, even before the appearance of a biofilm. This accumulation is concomitant with the conservation in the liquid (bioconvection). The biofilm appears during the growth phase of the bacterial population in an average concentration in the culture medium of about 10¹³ bacteria / m³. Then, we formed a continuous model that shows us the evolution of the environment of the bacteria. This model reproduced the bioconvection that was observed in the experiments and reveals its effect on the concentration of oxygen in the biological culture. Finally we built a hybrid continuous-discreet model that described the transition of the disconnected bacteria (nomads) through the connected bacteria that form a solid biofilm (sedentary). Each bacteria is considered as an individual particle. The model takes the contact forces under consideration, as well as the elastic forces that can settle between the bacteria when linked by the matrix. A minimum number of biological skills were used to form a model from the bacteria; the cellular division that allowed it to colonize the medium biological culture, the motility and the aerotaxi that explains its migration towards the liquid surface, the quorum sensing (QS) and the cellular differentiation that allows them to spend nomadic phenotype (motile) sedentary phenotype (producer of matrix). The dioxygen environment of the bacteria and its middle hydrodynamic properties are described by continuous fields. The model reproduces all the formation steps of an observed biofilm in our experiment and confirms the need of certain biological skills. It shows that the threshold of QS plays a major role in the morphology and biofilm formation kinetics. On the other hand, the rate of diocygen consumption by the bacteria does not seem to have any significant role. Finally, we established that the bioconvection reacts as a retardant in the biofilm formation.

A película adquirida (PA) é um filme formado pela adsorção seletiva de proteínas, glicoproteínas e lipídeos à superfície dentária. A presença de proteínas na PA forma uma interface protetora sobre a superfície do dente, participando em todos os eventos interfaciais que ocorrem na cavidade bucal, tais como des- e remineralização, lubrificação das superfícies dos dentes, e aderência bacteriana. Com o advento da proteômica, tem havido um aumento considerável no conhecimento acerca do perfil proteico de PAs adquiridas formadas sobre o esmalte dentário, em diferentes situações, mas nenhum trabalho até o momento descreveu o perfil proteômico de PAs formadas sobre a dentina. Este estudo foi pioneiro em comparar o perfil proteico de PAs formadas in situ sobre o esmalte e a dentina, nos tempos de 10 minutos e 2 horas, utilizando análise proteômica quantitativa livre de marcadores. Os experimentos foram realizados por três dias consecutivos. Em cada dia, os 9 voluntários receberam profilaxia dentária e em seguida utilizaram um aparelho vestibular com 6 blocos de esmalte e 6 de dentina humanos por 10 minutos ou 2 horas. Após esses períodos, a PA formada era coletada com auxílio de um papel filtro de eletrodos embebido em ácido cítrico 3%. Para as análises foi realizado um pool com os papéis dos 9 voluntários de todos os dias, para cada substrato e tempo de formação. Após a extração e digestão das proteínas, a separação dos peptídeos foi realizada por nano-HPLC (nano-Cromatografia Líquida de Alta Performace), interligada a um espectrômetro de massa (nLC-ESI-MS/MS). Os dados MS/MS obtidos foram processados e pesquisados em bancos de dados de proteínas humanas (UniProt e TrEMBL), utilizando o algoritmo SEQUEST no software Proteome Discoverer 1.3. Para a PA formada sobre o esmalte, foram identificadas 160 e 64 proteínas, nos tempos de formação de 10 minutos e 2 horas, respectivamente. Os respectivos números de proteínas identificadas para a dentina foram 86 e 52, respectivamente. Nos tempos de 10 minutos e 2 horas, respectivamente, 25 e 11 proteínas foram comuns a ambos os substratos e foram submetidas à quantificação livre de marcadores (SIEVE), revelando que a maioria das proteínas com diferença de expressão entre os dois substratos teve sua expressão aumentada na dentina. Foram identificadas ainda, no tempo de 10 minutos de formação da PA, 135 e 61 proteínas exclusivas ao esmalte ou à dentina, respectivamente. O número correspondente de proteínas exclusivas para o tempo de 2 horas foi de 53 e 41 proteínas, para o esmalte e dentina, respectivamente. Dentre as proteínas exclusivas da dentina, foram identificadas várias proteínas relacionadas ao complexo cálcio/calmodulina, assim como proteínas associadas à tumorigênese e à fosforilação/desfosforilação de proteínas. Em adição, muitas das proteínas identificadas no presente estudo, tanto para o esmalte quanto para a dentina, ainda não foram caracterizadas e, portanto, não têm função conhecida na PA. Sua caracterização e estudos funcionais futuros poderão trazer novos horizontes no entendimento da importância da PA para a proteção da estrutura dentária, bem como do papel da PA como sítio de biomarcadores para doenças bucais e sistêmicas. / The acquired pellicle (AP) is a film that results from selective adsorption of proteins, glicoproteins and lipids on the tooth surface. The presence of proteins in the AP forms a protective interface on the tooth surface that participates in all the surface events occurring in the oral cavity, such as de- and remineralization, lubrification of the tooth surfaces and bacterial adherence. With the advent of Proteomics, considerable increase in the knowledge of the protein profile of the AP formed on tooth enamel, under different circunstances, has been observed. However, so far the proteomic profile of the AP formed on dentin has not been described. This is the first study to compare the proteomic profile of APs formed in situ for 10 minutes and 2 hours, on enamel and dentin, using quantitative label-free proteomics. The experiments were conducted for 3 consecutive days. Each day, 9 volunteers were submitted to dental prophylaxis and in sequence wore a vestibular device containing 6 human enamel and 6 human dentin blocks for 10 minutes or 2 hours. After these periods, the PA formed was collected with an electrode filter paper soaked in 3% citric acid. The papers from the 9 volunteers, for each substrate and time of pellicle formation were pooled and used for analysis. After protein extraction and digestion, peptides were separated by nano-HPLC (High-performance liquid chromatography) coupled to a mass spectrometer (nLC-ESI- MS/MS). The obtained MS/MS spectra were searched against human protein databases (UniProt and TrEMBL) using SEQUEST algorithm in Proteome Discoverer 1.3 software. For the AP formed on enamel, 160 and 64 proteins were identified for the times of pellicle formation of 10 minutes and 2 hours, respectively. The respective numbers of identified proteins for dentin were 86 and 52, respectively. For the times of 10 minutes and 2 hours, respectively, 25 and 11 proteins were common to both substrates. They were submitted to label-free quantification, which revealed that most of the proteins with differential expression were overexpressed in the dentin. For APs formed for 10 minutes, 135 and 61 proteins were identified exclusively for enamel or dentin, respectively. The corresponding number for the 2-hour APs was 53 and 41 proteins, respectively. Among the proteins identified exclusively in dentin, many proteins related with calcium/calmodulin complex, as well as proteins associated with tumorigenesis and protein phosphorylation/dephosphorylation were found. In addition, many of the identified proteins, both for enamel and dentin, remain uncharacterized and, therefore have no described function in the AP. In the future, their characterization and functional studies might open new avenues for the understanding of the importance of the AP for the protection of the dental structure, as well as for the use of the AP as a site for biomarkers of oral and systemic diseases.

Os peptídeos da estaterina (DR9) e da histatina 3 (RR14), que ocorrem naturalmente na película in vivo, amplificam o efeito inibitório do crescimento de cristais de hidroxiapatita, função relacionada à remineralizarão do esmalte e formação de cálculos dentários. A hipótese da duplicação/hibridação de domínios funcionais dos peptídeos DR9 da estaterina e RR14 da histatina 3 foi testada. Para isto, os peptídeos peptidomiméticos (DR9-DR9, DR9-RR14), além deles individualmente e suas proteínas intactas (DR9, RR14, estaterina e histatina 3) foram estudados em sete concentrações diferentes para avaliar o efeito da inibição do crescimento de cristais de hidroxiapatita. Foi utilizado um ensaio colorimétrico de microplaca para quantificar o crescimento de cristais de hidroxiapatita. As experiências foram feitas em triplicata e a concentração inibitória (IC50) foi estabelecida para cada grupo. A IC50 foi calculada para todos os peptídeos e proteínas testados. A histatina 3 e o RR14 não atingiram o valor de IC50. O DR9- RR14 atingiu o valor de IC50 a 3,80 M. Como esperado, DR9 e DR9-DR9 demonstraram um efeito inibitório significativo na atividade de crescimento de cristais, atingindo o valor de IC50 a 2,82 M e 1,07 M, respectivamente. A estaterina atingiu o valor de IC50 a 2,50 M. Na análise estatística, foram aplicados os testes ANOVA e Student-Newman-Keuls para comparações por pares, para comparar os valores entre os grupos. O DR9-DR9 amplificou o efeito inibitório do crescimento de cristais de hidroxiapatita quando comparado com DR9 único (p <0,05), demonstrando que a multiplicação do domínio funcional é uma forte tendência evolutiva da proteína. De forma interessante, o peptídeo híbrido DR9-RR14 demonstrou um efeito inibitório intermediário quando comparado com outros dois grupos: DR9 único e DR9-DR9. Este estudo utilizou a abordagem peptidomimética para investigar uma via potencial de evolução da proteína relacionada com a duplicação/hibridação dos constituintes peptídicos naturais da película adquirida de esmalte. O conhecimento obtido por meio dos resultados deste trabalho pode fornecer uma base para o desenvolvimento de peptídeos sintéticos para uso terapêutico, tanto contra cárie dentária, como para a doença periodontal. / The statherin and histatin 3 peptides (DR9 and RR14 respectively), which occur naturally in the film in vivo, amplify the inhibitory effect for the growth of hydroxyapatite crystals, a function related to remineralization of the enamel and formation of dental calculi. The hypothesis of duplication/hybridization of functional domains of the DR9 peptides of the statherin and RR14 of histatin 3 was tested. For this, the peptidomimetic peptides (DR9-DR9, DR9-RR14), in addition to them individually and their intact proteins (DR9, RR14, statherin and histatin 3) were studied at seven different concentrations to evaluate the effect of growth inhibition of hydroxyapatite crystals. A colorimetric assay of microplate was used to quantify the growth of hydroxyapatite crystals. The experiments were done in triplicate and the inhibitory concentration (IC50) was established for each group. The IC50 was calculated for all peptides and proteins tested. Histatin 3 and RR14 did not reach the IC50 value. DR9-RR14 reached the IC50 value at 3.80 M. As expected, DR9 and DR9-DR9 demonstrated a significant inhibitory effect on crystal growth activity, reaching the IC50 value at 2.82 M and 1.07 M, respectively. Statherin reached the IC50 value at 2.50 M. ANOVA and Student-Newman-Keuls tests for paired comparisons were applied to compare the values between the groups. DR9-DR9 amplified the inhibitory effect of hydroxyapatite crystal growth when compared to single DR9 (p <0.05), demonstrating that the multiplication of the functional domain is a strong protein evolution pathway. Interestingly, the hybrid peptide DR9-RR14 demonstrated an intermediate inhibitory effect when compared to other two groups: single DR9 and DR9-DR9. This study utilized the peptidomimetic approach to investigate a potential pathway of protein evolution related to duplication/hybridization of the natural peptidic constituents of the acquired enamel film. The knowledge obtained through the results of this work can provide a basis for the development of synthetic peptides for therapeutic use, both against dental caries and for periodontal disease.

EPECa tem sido identificada como o principal agente de diarreia aguda em crianças de países em desenvolvimento. Biofilmes são estruturas bacterianas envoltas por uma matriz de exopolissacarídeos. O objetivo deste estudo foi verificar a influência de diferentes condições de cultivo na formação de biofilme por EPECa isoladas de crianças com diarreia e pesquisar a presença de genes relacionados com a formação de biofilme. As sequências genéticas dos genes csgA, crl, fimH e bcsA foram pesquisadas através da PCR e verificadas em 6 (26%), 23 (100%), 22 (95,6%) e 23 (100%) das amostras respectivamente. A formação de biofilme em placas de MTP por EPECa foi melhor evidenciada em meio LB sem sal a 26 °C e em caldo E. coli a 37 °C, tanto em número de amostras quanto em intensidade da formação de biofilme. As microscopias confocal e de varredura propiciaram uma análise qualitativa de microcolônias e pilares, além de EPS respectivamente. As condições de cultivo devem ser usadas com precaução para realmente aferir a capacidade de formação de biofilme por amostras de EPECa. / aEPEC has been identified as the main agent of acute diarrhea in children in developing countries. Biofilms are bacterial structures which are surrounded by a matrix of exopolysaccharides. The aim of this study was investigate the influence of different culture conditions on biofilm formation by 23 aEPEC strains isolated from children with diarrhea and search for the presence of genes related to biofilm formation. The genetic sequences of the csgA, crl, fimH and bcsA genes were screened by PCR and were found in 6 (26%), 23 (100%) 22 (95.6%) and 23 (100%) of the strains respectively. Biofilm formation in MTP plates for aEPEC strains was better evidenced in LB medium without NaCl at 26 ° C and in E. coli broth at 37 ° C, both in number and intensity of biofilm formation. The confocal and scanning electron microscopy provided a qualitative analysis of structures such as microcolonies and pillars, and respectively EPS. The growing conditions should be used with caution to measure the real ability of biofilm formation by strains of aEPEC.

O objetivo deste trabalho foi analisar as mudanças no perfil protéico em películas adquiridas formadas in situ, em diferentes tempos, sobre o esmalte e sobre a dentina humana após a exposição a dois tipos de ácido: lático e cítrico. Foram utilizados 162 blocos de esmalte e 162 blocos de dentina humana (3X3 mm). Os experimentos foram realizados em três dias consecutivos. Em cada dia, os voluntários (n=9) utilizavam um dispositivo mandibular contendo 6 blocos de esmalte e 6 blocos de dentina. Na sequência, os voluntários permaneceram com o dispositivo na cavidade bucal por 10 ou 120 minutos para formação de película adquirida. Os blocos foram então imersos em ácido cítrico (1%, pH 2,5) ou ácido lático (0,1 M pH 4,8) ou água deionizada por 20 segundos. Na sequência, a película foi removida com um papel de filtro umedecido em ácido cítrico a 3%. Este procedimento foi repetido por mais dois dias adicionais e foi confeccionado um pool com os papeis de filtro obtidos dos 9 voluntários, para cada tipo de substrato, tempo de coleta e meio de imersão. Após extração das proteínas, as mesmas foram submetidas à cromatografia líquida de fase reversa interligada a um espectrômetro de massas (nLC-ESI-MS/MS). Os dados obtidos de MS/MS foram processados e submetidos conjuntamente ao programa SEQUEST [Proteome Discoverer 1.3 (Thermo Scientific)]. As buscas foram feitas utilizando-se os bancos de dados SWISS-PROT e TrEMBL. Para o esmalte, a taxa de identificação de proteínas foi baixa (13 proteínas no total). Já para a dentina, a taxa de identificação de proteínas foi maior (223 proteínas no total), sendo que a exposição ao ácido cítrico reduziu dramaticamente o número de proteínas identificado, o que não aconteceu para o ácido lático. Proteínas ácido-resistentes foram identificadas tanto para o esmalte quanto para a dentina, destacando-se as queratinas e as mucinas, respectivamente. Estas proteínas, ou os peptídeos oriundos delas responsáveis pelo efeito protetor, são candidatas a serem utilizadas para o enriquecimento de produtos odontológicos, visando à prevenção da cárie e erosão dentária. / The purpose of this work was to analyze changes in protein profile in the acquired pellicle formed on enamel and dentine at different times in situ after exposure to lactic and citric acids. Enamel (n=162) and dentin (n=162) blocks (3X3 mm) were be used. The experiments were conducted on three consecutive days. Each day, volunteers (n = 9) used a mandibular device containing 6 blocks of enamel and 6 of human dentin. After the volunteers remained with the device in the oral cavity for 10 or 120 minutes in order to allow the formation of the acquired pellicle, the blocks were immersed in citric acid (1%, pH 2.5) or lactic acid (0.1 M, pH 4,8) or deionized water for 20 seconds. Following, the pellicle was collected with an electrode filter paper soaked in 3% citric acid. This procedure was repeated for two additional days and \'pools\' with the filter papers obtained from the 9 volunteers for each type of substrate, sampling time and type of acid were made. After extraction, proteins were subjected to reverse phase liquid chromatography coupled to mass spectrometry (nLC-ESI-MS/MS). MS/MS data were processed and submitted to SEQUEST software [Proteome Discoverer 1.3 (Thermo Scientific)]. Searches were done using SWISS-PROT and TrEMBL databases. The rate of protein identification was low for enamel (13 proteins in total). As for dentin, the rte of protein identification was higher (223 proteins in total). Exposure to citric acid dramatically reduced the number of identified proteins, what did not occur for lactic acid. Acid-resistant proteins, especially keratins and mucins were identified for enamel and dentin, respectively. These proteins, or the peptides originated from them that are responsible for the protective effect, are candidates to be used for the enrichment of dental products, aiming to prevent dental caries and erosion.

O objetivo deste trabalho foi analisar as mudanças no perfil protéico em películas adquiridas formadas in situ, em diferentes tempos, sobre o esmalte e sobre a dentina humana após a exposição a dois tipos de ácido: lático e cítrico. Foram utilizados 162 blocos de esmalte e 162 blocos de dentina humana (3X3 mm). Os experimentos foram realizados em três dias consecutivos. Em cada dia, os voluntários (n=9) utilizavam um dispositivo mandibular contendo 6 blocos de esmalte e 6 blocos de dentina. Na sequência, os voluntários permaneceram com o dispositivo na cavidade bucal por 10 ou 120 minutos para formação de película adquirida. Os blocos foram então imersos em ácido cítrico (1%, pH 2,5) ou ácido lático (0,1 M pH 4,8) ou água deionizada por 20 segundos. Na sequência, a película foi removida com um papel de filtro umedecido em ácido cítrico a 3%. Este procedimento foi repetido por mais dois dias adicionais e foi confeccionado um pool com os papeis de filtro obtidos dos 9 voluntários, para cada tipo de substrato, tempo de coleta e meio de imersão. Após extração das proteínas, as mesmas foram submetidas à cromatografia líquida de fase reversa interligada a um espectrômetro de massas (nLC-ESI-MS/MS). Os dados obtidos de MS/MS foram processados e submetidos conjuntamente ao programa SEQUEST [Proteome Discoverer 1.3 (Thermo Scientific)]. As buscas foram feitas utilizando-se os bancos de dados SWISS-PROT e TrEMBL. Para o esmalte, a taxa de identificação de proteínas foi baixa (13 proteínas no total). Já para a dentina, a taxa de identificação de proteínas foi maior (223 proteínas no total), sendo que a exposição ao ácido cítrico reduziu dramaticamente o número de proteínas identificado, o que não aconteceu para o ácido lático. Proteínas ácido-resistentes foram identificadas tanto para o esmalte quanto para a dentina, destacando-se as queratinas e as mucinas, respectivamente. Estas proteínas, ou os peptídeos oriundos delas responsáveis pelo efeito protetor, são candidatas a serem utilizadas para o enriquecimento de produtos odontológicos, visando à prevenção da cárie e erosão dentária. / The purpose of this work was to analyze changes in protein profile in the acquired pellicle formed on enamel and dentine at different times in situ after exposure to lactic and citric acids. Enamel (n=162) and dentin (n=162) blocks (3X3 mm) were be used. The experiments were conducted on three consecutive days. Each day, volunteers (n = 9) used a mandibular device containing 6 blocks of enamel and 6 of human dentin. After the volunteers remained with the device in the oral cavity for 10 or 120 minutes in order to allow the formation of the acquired pellicle, the blocks were immersed in citric acid (1%, pH 2.5) or lactic acid (0.1 M, pH 4,8) or deionized water for 20 seconds. Following, the pellicle was collected with an electrode filter paper soaked in 3% citric acid. This procedure was repeated for two additional days and \'pools\' with the filter papers obtained from the 9 volunteers for each type of substrate, sampling time and type of acid were made. After extraction, proteins were subjected to reverse phase liquid chromatography coupled to mass spectrometry (nLC-ESI-MS/MS). MS/MS data were processed and submitted to SEQUEST software [Proteome Discoverer 1.3 (Thermo Scientific)]. Searches were done using SWISS-PROT and TrEMBL databases. The rate of protein identification was low for enamel (13 proteins in total). As for dentin, the rte of protein identification was higher (223 proteins in total). Exposure to citric acid dramatically reduced the number of identified proteins, what did not occur for lactic acid. Acid-resistant proteins, especially keratins and mucins were identified for enamel and dentin, respectively. These proteins, or the peptides originated from them that are responsible for the protective effect, are candidates to be used for the enrichment of dental products, aiming to prevent dental caries and erosion.

A película adquirida (PA) é um filme formado pela adsorção seletiva de proteínas, glicoproteínas e lipídeos à superfície dentária. A presença de proteínas na PA forma uma interface protetora sobre a superfície do dente, participando em todos os eventos interfaciais que ocorrem na cavidade bucal, tais como des- e remineralização, lubrificação das superfícies dos dentes, e aderência bacteriana. Com o advento da proteômica, tem havido um aumento considerável no conhecimento acerca do perfil proteico de PAs adquiridas formadas sobre o esmalte dentário, em diferentes situações, mas nenhum trabalho até o momento descreveu o perfil proteômico de PAs formadas sobre a dentina. Este estudo foi pioneiro em comparar o perfil proteico de PAs formadas in situ sobre o esmalte e a dentina, nos tempos de 10 minutos e 2 horas, utilizando análise proteômica quantitativa livre de marcadores. Os experimentos foram realizados por três dias consecutivos. Em cada dia, os 9 voluntários receberam profilaxia dentária e em seguida utilizaram um aparelho vestibular com 6 blocos de esmalte e 6 de dentina humanos por 10 minutos ou 2 horas. Após esses períodos, a PA formada era coletada com auxílio de um papel filtro de eletrodos embebido em ácido cítrico 3%. Para as análises foi realizado um pool com os papéis dos 9 voluntários de todos os dias, para cada substrato e tempo de formação. Após a extração e digestão das proteínas, a separação dos peptídeos foi realizada por nano-HPLC (nano-Cromatografia Líquida de Alta Performace), interligada a um espectrômetro de massa (nLC-ESI-MS/MS). Os dados MS/MS obtidos foram processados e pesquisados em bancos de dados de proteínas humanas (UniProt e TrEMBL), utilizando o algoritmo SEQUEST no software Proteome Discoverer 1.3. Para a PA formada sobre o esmalte, foram identificadas 160 e 64 proteínas, nos tempos de formação de 10 minutos e 2 horas, respectivamente. Os respectivos números de proteínas identificadas para a dentina foram 86 e 52, respectivamente. Nos tempos de 10 minutos e 2 horas, respectivamente, 25 e 11 proteínas foram comuns a ambos os substratos e foram submetidas à quantificação livre de marcadores (SIEVE), revelando que a maioria das proteínas com diferença de expressão entre os dois substratos teve sua expressão aumentada na dentina. Foram identificadas ainda, no tempo de 10 minutos de formação da PA, 135 e 61 proteínas exclusivas ao esmalte ou à dentina, respectivamente. O número correspondente de proteínas exclusivas para o tempo de 2 horas foi de 53 e 41 proteínas, para o esmalte e dentina, respectivamente. Dentre as proteínas exclusivas da dentina, foram identificadas várias proteínas relacionadas ao complexo cálcio/calmodulina, assim como proteínas associadas à tumorigênese e à fosforilação/desfosforilação de proteínas. Em adição, muitas das proteínas identificadas no presente estudo, tanto para o esmalte quanto para a dentina, ainda não foram caracterizadas e, portanto, não têm função conhecida na PA. Sua caracterização e estudos funcionais futuros poderão trazer novos horizontes no entendimento da importância da PA para a proteção da estrutura dentária, bem como do papel da PA como sítio de biomarcadores para doenças bucais e sistêmicas. / The acquired pellicle (AP) is a film that results from selective adsorption of proteins, glicoproteins and lipids on the tooth surface. The presence of proteins in the AP forms a protective interface on the tooth surface that participates in all the surface events occurring in the oral cavity, such as de- and remineralization, lubrification of the tooth surfaces and bacterial adherence. With the advent of Proteomics, considerable increase in the knowledge of the protein profile of the AP formed on tooth enamel, under different circunstances, has been observed. However, so far the proteomic profile of the AP formed on dentin has not been described. This is the first study to compare the proteomic profile of APs formed in situ for 10 minutes and 2 hours, on enamel and dentin, using quantitative label-free proteomics. The experiments were conducted for 3 consecutive days. Each day, 9 volunteers were submitted to dental prophylaxis and in sequence wore a vestibular device containing 6 human enamel and 6 human dentin blocks for 10 minutes or 2 hours. After these periods, the PA formed was collected with an electrode filter paper soaked in 3% citric acid. The papers from the 9 volunteers, for each substrate and time of pellicle formation were pooled and used for analysis. After protein extraction and digestion, peptides were separated by nano-HPLC (High-performance liquid chromatography) coupled to a mass spectrometer (nLC-ESI- MS/MS). The obtained MS/MS spectra were searched against human protein databases (UniProt and TrEMBL) using SEQUEST algorithm in Proteome Discoverer 1.3 software. For the AP formed on enamel, 160 and 64 proteins were identified for the times of pellicle formation of 10 minutes and 2 hours, respectively. The respective numbers of identified proteins for dentin were 86 and 52, respectively. For the times of 10 minutes and 2 hours, respectively, 25 and 11 proteins were common to both substrates. They were submitted to label-free quantification, which revealed that most of the proteins with differential expression were overexpressed in the dentin. For APs formed for 10 minutes, 135 and 61 proteins were identified exclusively for enamel or dentin, respectively. The corresponding number for the 2-hour APs was 53 and 41 proteins, respectively. Among the proteins identified exclusively in dentin, many proteins related with calcium/calmodulin complex, as well as proteins associated with tumorigenesis and protein phosphorylation/dephosphorylation were found. In addition, many of the identified proteins, both for enamel and dentin, remain uncharacterized and, therefore have no described function in the AP. In the future, their characterization and functional studies might open new avenues for the understanding of the importance of the AP for the protection of the dental structure, as well as for the use of the AP as a site for biomarkers of oral and systemic diseases.