L’objectif de cette étude est d’étudier le rôle de la salive dans la libération des molécules odorantes, par deux approches, in vitro et in vivo. L’effet des protéines salivaires sur la libération de 10 molécules odorantes (5 esters et 5 cétones, de longueur de chaîne hydrophobe variable) a été étudié in vitro dans des systèmes modèles composés de salives artificielles et humaine. Les salives artificielles contiennent les protéines majoritairement présentes dans la salive (mucine et alpha-amylase), seules et en mélange. Les quantités de chaque molécule odorante présentes dans la phase gazeuse à l’équilibre thermodynamique ont été mesurées par une analyse headspace en mode statique couplée à la chromatographie en phase gazeuse (SH-GC). Les coefficients de partage entre l’air et chacun des systèmes modèles ont été calculés pour chacune des molécules. Cette approche in vitro nous a permis de démontrer une diminution des coefficients de partage air/salive artificielle en présence de mucine et d’alpha-amylase, par un effet hydrophobe. Aucun effet cumulatif n’est observé lorsque les deux protéines sont mises ensemble en solution. En présence de salive humaine, une diminution des coefficients de partage est également observée, les esters étant plus affectés par la présence de salive humaine que les cétones. Cette observation est due à une activité des estérases de la salive, qui augmente avec l’hydrophobicité des esters. La libération in vivo du propanoate d’éthyle et de l’hexanoate d’éthyle a été suivie sur 10 sujets par spectrométrie de masse à ionisation chimique à pression atmosphérique (APCI-MS) dans des conditions physiologiques différentes : au repos, après stimulation et après élimination du film salivaire résiduel. La salive de chaque sujet a été caractérisée dans les différentes conditions physiologiques testées. De grandes variations de flux, viscosité et de composition salivaire ont été mises en évidence entre les sujets, ainsi qu’entre les conditions physiologiques pour un même sujet. Les différences observées sur les paramètres de libération in vivo des molécules odorantes sont discutées en regard de ces paramètres physiologiques. Nous avons ainsi observé qu’une viscosité salivaire élevée diminue la quantité de molécules odorantes libérées sur un temps donné. Dans le même temps, la présence d’une quantité importante d’alpha amylase dans la salive augmente de façon significative le temps de libération de la molécule la plus hydrophobe, l’hexanoate d’éthyle. Nous avons ainsi mis en évidence que la rétention des molécules hydrophobes par les protéines salivaires peut induire une modification de leur cinétique de libération en conditions réelles de consommation et pourrait intervenir dans la persistance aromatique. / The aim of this work is to give a deeper understanding of the impact of the salivary composition on aroma release, by two approaches, an in vitro and an in vivo approach. The impact of salivary proteins on the release of 10 aroma compounds (5 esters and 5 ketones, varying in their hydrophobic chain length) was first investigated by in vitro model systems composed of artificial and human saliva. Artificial salivas were composed of the main salivary proteins, mucins and alpha amylase, alone and in mixture.The amount of aroma released in the vapor phase at equilibrium was analyzed by Static Headspace Gas Chromatography analysis. Air/system partition coefficients have been calculated. This in vitro approach allowed us to demonstrate the ability of both mucin and alpha-amylase to decrease the release of aroma compounds by hydrophobic effect (increase of retention with aroma hydrophobicity). Interestingly, no cumulative effect was observed when both proteins were mixed together in solution. The release of ketones in presence of human saliva is lower than in water and slightly higher than in the presence of artificial saliva. Esters are more affected by the presence of human saliva than ketones. This observation is due to an esterase activity of saliva, which increases with the hydrophobicity of esters. The in vivo release of ethyl propanoate and ethyl hexanoate was followed on ten subjects by Atmospheric Pressure Chemical Ionization mass spectrometry (APCI-MS) under different physiological conditions: at rest, after stimulation and after removing the superficial salivary coat. The saliva was characterized for each subject and each physiological condition. Great variations were observed between the subjects on the salivary flow, viscosity, composition and for each subject between the physiological conditions. The differences observed on in vivo release parameters are discussed as a function of physiological parameters. We observed that subjects with a more viscous saliva present a lower amount of aroma released. The presence of higher amounts of alpha-amylase increased the time needed to release the more hydrophobic compound, ethyl hexanoate. Our results suggest that the retention of hydrophobic aroma compounds by salivary proteins induces a modification of the kinetics of aroma release in real consumption conditions, and could be responsible for aroma persistence.

Ce travail de thèse vise à mieux comprendre l'effet de l'environnement buccal sur le comportement d'une bactérie orale probiotique, Streptococcus salivarius K12. La croissance et la maintenance de S. salivarius K12 ont tout d'abord été caractérisées dans une salive artificielle complémentée (CAS) conçue pour l'étude. Dans ce milieu, cette bactérie démontre un taux de croissance élevé et un temps de latence court, mais elle ne produit pas de bactériocines actives. La survie de S. salivarius K12 en phase stationnaire est, en revanche, affectée dans le milieu CAS. Ce phénomène est expliqué par une synthèse moindre des protéines impliquées dans le métabolisme énergétique, dont celui du glycogène. Toutefois, malgré une sensibilité accrue en phase stationnaire, le milieu CAS permet la croissance et la maintenance de S. salivarius K12. Les effets de plusieurs facteurs environnementaux spécifiques de la bouche, sur S. salivarius K12, ont été déterminés en milieu CAS. Ainsi, l'apport de saccharose, conduit à une dégradation de la viabilité. Des enzymes, ajoutées à leur concentration physiologique, affectent également les cellules bactériennes. Le lysozyme accroît la mortalité de S. salivarius par son action sur la paroi bactérienne. La peroxidase améliore sa viabilité en diminuant le potentiel redox du milieu. Le rôle clé du potentiel redox sur S. salivarius K12 est confirmé par l'impact négatif de l'injection d'air enrichi à 5% de CO2, qui accroît le potentiel redox. Enfin, l'amylase a démontré un rôle à la fois positif (augmentation de la biomasse) et négatif (diminution du taux de croissance). En conséquence, les études impliquant des bactéries orales se doivent de prendre en compte ces facteurs environnementaux influant sur les l'état physiologique bactérien. La mise en place de cultures continues respectant les variations de flux salivaire et permettant l'apport périodique de nutriments, tout en combinant l'ensemble des conditions environnementales identifiées précédemment, a permis de simuler la dynamique des conditions buccales. Les résultats démontrent que S. salivarius K12 est bien adapté à ces conditions de culture. Les cellules sont capables de se maintenir à un niveau de cultivabilité constant, malgré la carence nutritionnelle et le lessivage auxquels elles sont soumises. Certains mécanismes moléculaires expliquant cette adaptation ont été caractérisés : activation des voies d'utilisation de sources de carbone alternatives, stockage de l'énergie, augmentation de la compétence génétique naturelle. Finalement, ces travaux ont permis d'identifier certains mécanismes permettant à Streptococcus salivarius K12 de s'adapter à l'environnement buccal, grâce à la mise en place de méthodes d'étude in vitro du comportement des bactéries orales. / This thesis aims to better understand the effect of oral environmental conditions on the behaviorof the probiotic bacteria Streptococcus salivarius K12. Growth and maintenance of S. salivarius K12 have been characterized in a complemented artificial saliva (CAS), designed for this study. In this medium, S. salivarius demonstrated highspecific growth rate and low lag time, but it did not produce active bacteriocins. However, the survival of S. salivarius K12 during stationary phase was affected during fermentation in CAS medium. This was mainly explained by a reduced synthesis of proteins involved in energy and glycogen metabolisms. Thus, despite an increased sensitivity in stationary phase, the "complemented artificial saliva" allowed the growth and maintenance of S. salivarius K12. The effects of several environmental oral factors on S. salivarius K12 were determined in complemented artificial saliva. Adding sucrose decreased cellular viability. Enzymes added to their physiological concentration also affected the bacteria. Lysozyme increased S. salivarius mortality by acting on cellular wall. Peroxidase enhanced viability, by reducing the redox potential. The key role of redox potential on S. salivarius K12 was confirmed by the negative impact of the injection of air containing 5% CO2, which increased redox potential. The amylase demonstrated both positive (biomass increase) and negative roles (reduced growth rate). Consequently, studies involving oral bacteria must integrate these environmental factors that affected the bacterial physiological state. Continuous cultures, taking into account the variations in salivary flow and the periodical supply of nutrients, and combining all environmental conditions previously identified, allowed simulating oral dynamic conditions. From our results, a good adaptation of S. salivarius K12 took place in these culture conditions. Cells were able maintaining a constant level of cultivability despite nutritional starvation and wash out. Some molecular mechanisms explaining this bacterial adaptation have been characterized: activation of alternative carbon sources pathways, energy storage, and increase of natural genetic competence. Finally, this work made it possible identifying some mechanisms used by Streptococcussalivarius K12 to adapt itself to the oral environment, through the establishment of in vitro methods for studying the behavior of oral bacteria.

The polymeric gel-forming mucins provide the structural framework of saliva and the mucus barriers that cover the mucosal surfaces of the alimentary canal. Dietary compounds may influence the barrier properties of these protective layers. The effects of green tea polyphenols, which have many health benefits but have low bioavailability and contribute to the astringency of green tea, on the structural properties of the mucins in the alimentary canal are investigated here. Using well characterised, highly purified salivary mucins MUC5B and MUC7, and porcine gastric mucins, the effects of the green tea polyphenol epigallocatechin-3-gallate (EGCG) on mucins were studied here. Using rate-zonal centrifugation coupled to agarose gel electrophoresis, atomic force microscopy and particle tracking microrheology, EGCG, at concentrations found in a cup of green tea, caused increased aggregation of MUC5B in human whole saliva, and increased aggregation and viscosity of purified MUC5B. It was revealed using recombinant proteins of the N- and C-terminal regions of MUC5B that EGCG had these effects by aggregating the terminal globular protein domains of MUC5B. In contrast, MUC5B trypsin-resistant high molecular weight glycopeptides were not aggregated by EGCG, demonstrating that the oligosaccharide-rich, highly-glycosylated regions of mucins are not involved in the EGCG-induced aggregation of mucins. EGCG also caused the majority of MUC7 in human whole saliva to aggregate, and purified MUC7 also showed substantial aggregation in the presence of EGCG.Porcine gastric mucins were also used in order to model human gastric mucins. First, the identity of the porcine gastric mucins was explored using tandem mass spectrometry and immunohistochemistry. This revealed that Muc5ac was expressed by the surface epithelium and was the prominent mucin in porcine gastric mucus. Muc6 was expressed by gastric submucosal glands, but was not a major component of the secreted mucus barrier. Porcine Muc5ac and Muc6 were shown to be aggregated by EGCG. These data demonstrate that mucins from both saliva and the stomach are substantially altered by EGCG. This may contribute to the astringency and low bioavailability of EGCG. In contrast, the green tea polyphenol epicatechin (EC) did not cause aggregation of salivary mucins or porcine gastric mucins, suggesting that the galloyl ring of EGCG (which is absent in EC) is important for its aggregation of mucins, and that EC has different mechanisms of astringency. The structure of the mucins in the alimentary canal was studied using Raman spectroscopy, Raman optical activity (ROA) and Tip-enhanced Raman spectroscopy (TERS). The secondary structure of the oligosaccharide-rich regions of mucins was shown to be largely disordered, with some contribution of poly-proline II helix. The N- and C-terminal regions of MUC5B were largely β-sheet in structure, with some disordered structure also present in the C-terminal region. Raman spectroscopy could reliably distinguish between MUC5B glycoforms, demonstrating the sensitivity of this technique to mucin glycosylation and secondary structure. The first TERS spectra along the length of a MUC5B chain are reported, and suggest that patterns may exist in the glycosylation of MUC5B. Therefore, Raman spectroscopies are novel tools that shed new light on mucin structure and in future may be useful for studying the changes to mucin structure during interactions, such as those with polyphenols.

A presente tese teve como objetivo realizar a descrição dos microrganismos que já foram isolados ou detectados em infecções endodônticas de dentes decíduos em pacientes infantis por meio de uma revisão sistemática, além de avaliar a composição bacteriana e a presença de genes de resistência a antibióticos em amostras de saliva (S), biofilme supragengival (SB), dentina (D) e câmara pulpar (RC) de dentes decíduos com infecções endodônticas. No Capítulo 1, realizou-se revisão sistemática em bancos de dados eletrônicos, tendo sido incluídos estudos clínicos que avaliaram presença de microrganismos em dentes decíduos com infecções endodônticas, por meio de análise microbiológica com cultivo ou de métodos moleculares. Foi realizada análise descritiva dos dados. A análise identificou 44 títulos, sendo revisados, na íntegra, 17 artigos. Foram selecionados 8 estudos clínicos, de acordo com os critérios de inclusão determinados. Por meio de busca manual, foram selecionados 2 artigos adicionais, totalizando 11 artigos excluídos desta revisão. Nos oito estudos clínicos incluídos na revisão sistemática, a identificação dos microrganismos envolvidos nas infecções endodônticas foi realizada por meio de várias técnicas como: cultura microbiológica, hibridização DNA-DNA, PCR e suas variações, clonagem, sequenciamento e pirossequenciamento, confirmando a diversidade de microrganismos envolvidos nas infecções endodônticas de dentes decíduos. A análise dos dados sugere que as infecções endodônticas em dentes decíduos são causadas por múltiplas combinações de espécies de micro-organismos, confirmando a sua natureza polibacteriana. No Capítulo 2, amostras de S, SB, D e RC foram coletadas de pacientes infantis com infecções endodônticas. O perfil das comunidades microbianas foram obtidos por meio da análise da região espaçadora intergênica relacionada aos genes 16S e 23S rRNA (PCR-RISA). Determinaram-se e índices de riqueza, dominância, índice de Shannon, índice de Chao-1 (alfa-diversidade) e análise multivariada de conglomerados (método UPGMA e índice de Similaridade de Bray-Curtis) e análise de coordenadas principais (PCoA) (beta-diversidade). Há um baixo grau de agrupamento entre as amostras de S, BS, D e RC, obtidas de um mesmo participante. Se presentes, os agrupamentos acontecem para sítios contíguos, mas com baixo percentual de similaridade. Amostras de um mesmo ecossistema obtidas de diferentes participantes abrigam comunidades bacterianas distintas, com baixa similaridade. Não parece haver uma relação entre a presença de um sinal/sintoma clínico e acréscimo no perfil de similaridade das comunidades bacterianas em RC. Não foram observadas diferenças estatisticamente significativas entre os índices de alfa-diversidade (riqueza, dominância, Shannon e Chao-1) entre S, SB, D e RC. O uso prévio de antibióticos não modificou os resultados de alfa diversidade ou de beta diversidade obtidos. No Capítulo 3, verificou-se a distribuição dos genes de resistência bacteriana aos principais grupos de antibióticos em S, SB, D, e RC dentes decíduos em pacientes infantis com infecções endodônticas e também de amostras de saliva dos responsáveis (R) por meio de PCR para os genes cfxA/cfxA2, blaTEM, blaZ, ampC, mecA, mefA, ermB, ermC, tetQ, tetM, tetW, linB, lsaB. Realizou-se análise estatística descritiva e análise multivariada de conglomerados (método UPGMA e índice de Similaridade de Bray-Curtis). Dos pacientes selecionados, 3/8 utilizaram antibiótico previamente à coleta. Nenhum gene de resistência foi observado em todos os ecossistemas de um mesmo participante. Os genes mais frequentemente detectados foram os genes de resistência à tetraciclina tetQ e tetW. Não foram detectados nas amostras os genes ampC, mecA, lnuB e lsaB. A presença simultânea de um gene em dois nichos ocorre em ecossistemas contíguos. Não se observa um comportamento uniforme quanto ao perfil de agrupamento de diferentes amostras de um mesmo participante, e nem entre as amostras de saliva do participante infantil (S) e seu responsável (R). Há múltiplos perfis de distribuição de genes de resistência a agentes antimicrobianos em amostras de ecossistemas bucais contíguos em um mesmo paciente portador de infecção endodôntica. A análise conjunta dos dados permite concluir que cada um dos ecossistemas da cavidade bucal de crianças portadoras de infecções endodônticas avaliado apresenta espécies bacterianas e fatores de virulência distribuídos de forma única e distintas, a partir de uma perspectiva de análise de diversidade. / This thesis aimed assessing information on the bacteria that were isolated/detected in teeth with endodontic infections from infant patients through a sistematic review of the literature. Furthermore, the bacterial composition and the presence of resistance genes to antimicrobial agents was determined in saliva (S), in supragengival biofilm (SB), in dentine (D) and in pulp cavity (RC) samples. In the Chapter 1, a sistematic review was conducted in electronic databasis. Clinical studies that evaluated the presence of microorganims in primary teeth with endodontic infections through culture and molecular methods were included. Fourty-four titles were selected and 17 articles were fully revised. Eight clinical studies were selected for data extraction. Two articles were included following the hand search. According to the data analysis, microbial identification was performed by culture, DNA-DNA hybridization, PCR, cloning and sequencing and next-generation sequencing methods. A high diversity in the microbial components identificated/detected was reported. Endodontic infections in primary teeth are polymicrobial, with a multi-species consortia. In the Chapter 2, the S, SB, D and RC samples were collected from infanti patients with endodontic infections. The ribossomal intergenic spacer analysis (PCR-RISA) for the 16S-23S rRNA genes interspacer region was employed to determine the bacterial fingerprint for each sample. Metrics for alfa and beta diversity were employed, such as richness, dominance, Shannon Index, Chao-1 Index, cluster analysis (UPGMA, Bray-Curtis Index) and principal coordenate analysis (PCoA). There was a low grouping profile for shared samples of S, BS, D and RC from the same participant. When detected, clustering behavior was observed for contiguous sites, with low percentual of similarity between them. Samples from the same site but from different subjects harboured distinct bacterial communities, with low similarity. No clinical sign/symptom was detected as a grouping factor for RC sample from different subjects. No statistical difference was detected for the alfa-diversity indexes among S, SB, D and RC. The previous exposition to antimicrobial agentes has no effect over the alfa- and beta-diversity indexes. In the Chapter 3, the distribution of bacterial genes for antimicrobial resistance in S, SB, D and RC was determined in samples from children with endodontic infections and their relatives (R) by PCR. The presence of the genes cfxA/cfxA2, blaTEM, blaZ, ampC, mecA, mefA, ermB, ermC, tetQ, tetM, tetW, linB, and lsaB was detecte in the samples. Descriptive statistical analysis and multivariate anlysis (cluster analyisis, UPGMA and Bray-Curtis similarity index) were carried out. Three out of 8 patients had antimicrobial agents previously to the apointment. No resistance gene was shared by all envirnments in the same participant. The most frequently detected genes were tetQ and tetW. The genes ampC, mecA, lnuB, and lsaB were not detected in any the samples. The same gene was detected only in two contiguous niches. Clustering analysis revealed no grouping pattern among the samples, despite they were or not from the same participant or his/her relative. Multiple profiles of resistance genes distribution were detected in the oral cavity samples from infant participants. The oral cavity in children with endodontic infection is a complex environment that harbours unique bacterial communities profiles and a distinct distribution of resitance genes to antimicrobial agents, considering an ecological perspective.

Orientador: Mary Marcondes / Banca:Wagner Luis Ferreira / Banca: Suely Regina Mogami Bomfim / Banca: Juliana Peloi Vides / Banca: Acácio Duarte Pacheco / Resumo: O presente estudo teve como objetivos investigar a presença de anticorpos anti-Leishmania spp. e anti-saliva de Lutzomyia spp., e a presença do DNA de Leishmania spp. no sangue periférico de 284 indivíduos atendidos em um hospital público de área endêmica para leishmaniose visceral (LV). Baseando-se nos resultados de sorologia e da reação em cadeia da polimerase (PCR), 21,1% dos indivíduos foram considerados expostos. A presença de anticorpos anti-Leishmania spp. e anti-saliva de Lutzomyia spp. foi observada em 20,8% e 37,7% dos indivíduos, respectivamente, observando-se uma associação significativa entre a presença dos dois anticorpos. Em 20,4% dos indivíduos verificou-se sororeatividade em ambos os testes, 17,3% apresentavam anticorpos anti-saliva de Lutzomyia spp. sem a presença de anticorpos anti-Leishmania spp. e um indivíduo apresentava somente anticorpos anti-Leishmania spp. sem a presença de anticorpos anti-saliva de flebotomíneo. Em apenas um indivíduo foi possível amplificar fragmento do DNA de Leishmania spp. no sangue periférico. Concluiu-se que a infecção por Leishmania spp. pode estar sendo subdiagnosticada em áreas endêmicas para LV e, portanto, sugere-se que indivíduos atendidos em hospitais sejam melhor avaliados quanto à possibilidade de infecção e posterior desenvolvimento da doença. Ainda, a presença de anticorpos anti-saliva de flebotomíneo pode ser um possível indicador de exposição ao vetor. / Abstract: The present study aimed to investigate the presence of anti-Leishmania spp. and anti-saliva of Lutzomyia spp. antibodies, and the presence of Leishmania spp. DNA in the peripheral blood of 284 people attended at a public hospital in an endemic area for visceral leishmaniasis (VL). Based on the results of serology and PCR, 21.1% of the people were considered exposed. The presence of antibodies anti-Leishmania spp. and anti-saliva of Lutzomyia spp. was observed in 20.8% and 37.7% of the individuals, respectively, with a significant association between the presence of both antibodies. In 20.4% of the people there was serum reactivity in both tests, 17.3% had antibodies anti-saliva of Lutzomyia spp. without the presence of anti-Leishmania spp. antibodies, while one individual had only anti-Leishmania spp. antibodies without the presence of antibodies against saliva of sand flies. In only one individual it was possible to amplify the DNA fragment of Leishmania spp. in peripheral blood. It was concluded that the infection by Leishmania spp. may be underdiagnosed in endemic areas for VL and, therefore, it is suggested that people referred to hospitals should be better evaluated for the possibility of infection and subsequent development of the disease. Furthermore, the presence of antibodies anti-sandfly saliva may be an indicator of exposure to vector. / Doutor

Magister Chirurgiae Dentium (MChD) / Background: This study aimed to determine the association between dimensional change and surface roughness (Ra) of Vitremer, Activa and Cention N after immersing them into two different media: acidic and artificial saliva media for the period of a year. Measurements were made at 10 time intervals during the observation period. Methodology: This was a quantitative and qualitative study. For the quantitative part, a total of 60 specimens were tested, 20 specimens for each material. The 20 specimens were further divided into 10 specimens. Ten were immersed in acidic media and the rest in saliva media. A measurement of the weight, height, and Ra was carried out as follows: day 0, day 1, day 2, day 7, day 21, day 28, day 60, day 90, day 180 and day 365. Scanning electron microscopy (SEM) was used to examine the surface of each material qualitatively pre and post immersion in the two media. For fluoride measurements, an additional five samples from each material were left suspended in the de-ionized water by the use of dental floss. The materials were moved to new specimen jars after the completion of day 1, 2, 3, 4, 5, 6, 7, 14, 21 and 28. All the specimen jars had been kept for the fluoride measurements. Results: Non-parametric tests were used to analyze the data. Linear regression analysis was used to measure the association between weight, height or surface roughness (Ra) and immersion time for a year. The result of this test showed that Vitremer had a significant association between the weight (p = 0.000), height (p = 0.007) and Ra (p = 0.001) when it was immersed in acidic media. On the other hand, when Vitremer was immersed in saliva media, only the weight variable showed a significant association (p = 0.002). For Cention N, significant association was found for only Ra when immersed in acidic media (p = 0.000). Finally, for Activa, all the studied associations; the weight, height and Ra in both media were found to be insignificant. For saliva media, there was a significant weight change between the three materials during all 10 periods of time (p = 0.000). In the first six months, Cention N demonstrated a significant increase in weight changes followed by Vitremer, then Activa. Yet, after a year, the difference between Cention N and Vitremer became insignificant and Activa showed the least weight changes. There was not a significant difference between the materials in terms of height and Ra measurements. The fluoride experiment was not successful due to technical issues during pH measurements of the collected solutions. For comparison of the studied parameters between the three materials, the Kruskal-Wallis test was used. In acidic media, there was a significant difference between the materials in term of weight change in 10 periods of time (p = 0.000). In particular, after a two month period, Cention N had the highest weight, followed by Vitremer and then by Activa. The difference between Vitremer and Activa became insignificant throughout the rest of the experimental time frame. All the height measurements between the three materials were found to be insignificant except for day 365 (p = 0.048), where both Activa and Cention N were found to be significantly higher than Vitremer. For the Ra comparison, in the first two weeks, particularly day 1, 7 and 14, Cention N had significantly the lowest Ra among the other materials. As the three materials aged in the acidic media (day 180), Vitremer had significantly the highest Ra values. Cention N showed higher Ra values than Activa; nonetheless this difference was not significant. The SEM images showed loss of some particles in all post-experimental images of the materials in acidic media. Vitremer showed the widest cracks with the loss of fillers. In saliva media, there was also loss of particles but to a lesser extent than in acidic media. Yet, the post-experimental image of Activa in saliva resembled the pre-experimental one. Conclusion: Within the limitations of the study, the best material to resist Ra from prolonged acidic attack was Activa followed by Cention N and then Vitremer. Except for Vitremer, no significant changes in the Ra of the other materials were detected when the three materials were immersed in saliva media in the long term. In acidic media Vitremer tended to lose weight and height faster than Cention N and Activa over a year. Cention N is the best material to resist dimensional change. However, in artificial saliva Vitremer gained water rapidly. Activa did not absorb a lot of water and did not reject a lot of water; Activa demonstrated good dimensional stability and this property may be beneficial when compared to the other two materials tested. The clinical significance of the study: All the materials studied were subjected to dimensional and Ra changes following long-term exposure to acidic substances, but the newer materials (Cention N and Activa) seemed to be more dimensionally stable and resistant to Ra changes than the older, well-known material (Vitremer). This may influence a clinician’s choice of restorative material for use in pediatric dentistry.

Patients with bulimia nervosa are at high risk for dental erosion. However, not all bulimic patients suffer from erosion, irrespective of the severity of their eating disorder. It is often speculated that differences in the saliva are important, however, little is known about salivary parameters in bulimic patients, particularly directly after vomiting. The aim of the clinical trial was to compare different salivary parameters of subjects suffering from bulimia with those of healthy controls. Twenty-eight subjects participated (14 patients with bulimia nervosa, 7 of them with erosion; 14 matched healthy controls). Resting and stimulated saliva of all participants was analysed as well as saliva collected from bulimic patients directly and 30 min after vomiting. Parameters under investigation were flow rate, pH, buffering capacity and the enzyme activities of proteases in general, collagenase, pepsin, trypsin, amylase, peroxidase, and lysozyme. Regarding flow rate, pH and buffering capacity only small differences were found between groups; buffering capacity directly after vomiting was significantly lower in bulimic subjects with erosion than in subjects without erosion. Differences in enzymatic activities were more pronounced. Activities of proteases, collagenase and pepsin in resting and proteases in stimulated saliva were significantly higher in bulimic participants with erosion than in controls. Peroxidase activity was significantly decreased by regular vomiting. Proteolytic enzymes seem to be relevant for the initiation and progression of dental erosion directly after vomiting, maybe by both hydrolysis of demineralized dentine structures as well as modulation of the pellicle layer. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Glucocorticosteroide (GC) spielen für viele endogene Prozesse im Organismus eine wichtige Rolle. Sie regulieren die Gluconeogenese sowie den Lipid- und Proteinstoffwechsel. Außerdem sind sie für die Stressregulierung über die Hypothalamus-Nebennierenrinden-Achse verantwortlich. Therapeutisch kommen die GCs wegen ihrer entzündungshemmenden Wirkung zum Einsatz und werden u.a. bei Asthma und Gelenkentzündungen angewandt. Diese Eigenschaft macht sie auch interessant für den Gebrauch im Sportbereich. Dort wird ihre Anwendung über die Weltantidopingagentur reguliert. Ihr oraler, intramuskulärer, intravenöser und rektaler Gebrauch ist im Wettkampf verboten. Diese Einschränkung bzgl. des Applikationszeitraumes und des Applikationsweges erschwert die diagnostische Aussagekraft von Routinedopingproben, welche im Urin durchgeführt werden. Ein Grenzwert von 30 ng/ml soll einen legalen Gebrauch von einem Missbrauch abgrenzen. Die endogenen Glucocorticosteroide stellen hierbei jedoch einen Graubereich dar. Endogen wird Cortisol in einem zirkadianen Rhythmus produziert und die Produktion ist stressinduziert. Somit kommt es zu ausgeprägten intra- und interindividuellen Streuungen der endogenen Produktion. Dadurch bedingt ist eine Abgrenzung der endogenen Produktion von einer legalen Anwendung bzw. einem Missbrauch im Rahmen der Dopingrichtlinien im Urin nicht möglich. Speziell für den Nachweis von endogenen Substanzen ist es wichtig, eine Methode zu finden, mit der es möglich ist, die endogene Produktion von einer exogenen Bezugsquelle abzugrenzen. Dabei haben sich zwei Wege als hilfreich herausgestellt. Zum einen, wenn die Differenzierung nicht an Hand von Absolutkonzentrationen sondern durch die Anwendung von Analytverhältnissen durchgeführt wird. Zum anderen, wenn zusätzliche Untersuchungen im Speichel oder Haar durchgeführt werden. Haar- und Speichelproben zählen zu den ergänzenden Matrizes der Routineuntersuchungsmedien Urin und Blut und werden bereits in vielen forensischen und klinischen Laboren für diagnostische Fragestellungen verwendet. Diese Matrizes liefern wichtige Hinweise auf den akuten (Speichel) oder chronischen/ zurückliegenden (Haar) Gebrauch bzw. Missbrauch von Medikamenten und Drogen. Sowohl die Haar- als auch Speichelmatrix sollen den physiologisch aktiven Anteil von Substanzen im Blut widerspiegeln und somit korrektere Rückschlüsse auf deren Wirksamkeit zulassen. Das endogene Glucocorticosteroid Cortisol steht seit der Jahrtausendwende im Blickpunkt vieler Forschungen, welche sich mit dessen Bedeutung für die Stressantwort befassen und Cortisol u.a. im Speichel und Haar nachweisen. Auffällig ist dabei, dass die ersten Arbeiten fast ausschließlich mittels immunchemischen Nachweisverfahren erfolgten. Erst in den letzten fünf Jahren wurde vermehrt LC-MS/MS-Verfahren angewandt. Vorteil dieser ist, dass der Nachweis von Substanzen selektiv erfolgt und Kreuzreaktionen nicht stattfinden. Weiterhin ist es vorteilhaft, dass die Konzentrationen von mehreren Analyten mit einer Messung bestimmt werden können. So ist es zum Beispiel möglich Cortisol und andere Steroide, z.B. dem Cortison parallel nachzuweisen. Cortison spielt für die physiologische Wirkung der Glucocorticosteroide im Körper keine Rolle, da es selbst nicht biologisch aktiv ist. Deshalb wurde es in bisherigen Forschungen für diagnostische Aussagen nicht berücksichtigt. Mit Verwendung der LC-MS/MS-Technologie werden jedoch beide endogenen GCs zunehmend nebeneinander bestimmt. Bei der Betrachtung von unterschiedlichen Untersuchungsmedien ist auffällig, dass sich die Konzentrationsverhältnisse Cortisol zu Cortison unterscheiden. Entgegengesetzte Verhält-nisse werden ersichtlich, wenn die GC-Konzentrationen im Blut mit denen im Speichel bzw. Haar verglichen werden. Bisher wurden diese Beobachtungen mit der lokalen Wirksamkeit von Enzymen, welche die Corticosteroide ineinander umwandeln, erklärt. Im Rahmen der vorliegenden Dissertation wurde folgender Fragestellung für die Nachweisbarkeit der Glucocorticosteroide nachgegangen: „Wie hoch ist der Anteil der Plasmaproteinbindung der GCs im Blut und welche Rückschlüsse lassen sich daraus auf die Konzentrationsverschiebung innerhalb der einzelnen Matrizes ziehen?“ Basierend auf die einzelnen Teilprojekte wurden sowohl Plasmaproben als auch Speichel- und Haarproben hinsichtlich ihrer GC-Konzentrationen analysiert. Die Untersuchung von Kontrollproben ermöglichte es, Referenzwerte unter Normalbedingungen zu erheben. Die Ergebnisse aus den Projekten ergaben, dass die beiden endogen GCs Cortisol und Cortison in unterschiedlichen Konzentrationsverhältnissen in den Analysenmedien vorkommen: Plasma: Gesamtkonzentration F:E ca. 3:1 freie Konzentrationen F:E ca. 1:1 Speichel: F:E ca. 1:5 Haar: F:E ca. 1:3 Die Bestimmung der Plasmaproteinbindung (PPB) beider endogener GCs hat gezeigt, dass Cortisol mit ca. 96 % stärker an die Transportproteine CBG und Albumin bindet als Cortison mit ca. 85 %. Dies führt dazu, dass sich die freien, nicht-proteingebundenen Konzentrationen angleichen und es zu einer Verhältnisverschiebung von Cortisol zu Cortison von 3:1 auf 1:1 kommt. Somit stehen vergleichbare Konzentrationen für die Inkorporation ins Haar bzw. die Diffusion in den Speichel zur Verfügung. Es konnte gezeigt werden, dass die freien Plasmakonzentrationen beider GC stark mit den Speichelkonzentrationen korrelieren. Cortisol aber unterproportional und Cortison überproportional vom Plasma in den Speichel übergeht. Dies kann mit zwei weiteren Mechanismen, welche während der Diffusion eine Rolle spielen, der unterschiedlichen Lipophilie und der Inaktivierung durch lokale Enzym-reaktionen, erklärt werden. Weiterhin wurde gezeigt, dass sich die Tagesrhythmik der GC-Produktion im Speichel abbilden lässt und eine starke Korrelation zwischen Cortison und Cortisol vorliegt. Mit Hilfe einer Grenzfunktion können endogene Referenzkonzentrationen definiert und Messdaten eingeordnet werden. Unter anderem wurde gezeigt, dass eine Hormonersatztherapie mit Hydrocortison zu einer Verschiebung der Metabolisierung und der PPB führt und somit ein Gebrauch/Missbrauch von GCs durch abweichende Konzentrationsverhältnisse nachweisbar ist. Speicheluntersuchungen während einer chronischen Stresssituation (Schwangerschaft) zeigen, dass die GC-Produktion stetig ansteigt und sich besonders die morgendlichen Werte unterscheiden. Um die tageszeitlichen und stressbedingten Schwankungen der GC-Produktion auszublenden und eine längere Zeitspanne zu betrachten, wurden zusätzlich Haarproben analysiert. In diesen wurde ein kontinuierlicher Anstieg der GCs in den proximalen Haarsegmenten nachgewiesen, was auf eine kontinuierlich erhöhte Inkorporation während der chronischen Stresssituation schließen lässt. Außerdem wurde gezeigt, dass die Haarkonzentrationen dem Auswascheffekt unterliegen und die nachweisbaren Konzentrationen geringer werden, je älter das Haar wird. Schlussfolgernd kann gesagt werden, dass beide Mechanismen (Einlagerung und Auswaschung) konkurrieren und deshalb Referenzdaten nur für das proximale Segment erhoben werden können. Für weitere Segmente sind die Auswirkungen der individuellen Einflüsse nicht mehr allgemeingültig kalkulierbar und nur noch intraindividuelle Vergleiche nach mehrmaliger Beprobung aussagekräftig. Sind die Effekte der verstärkten Inkorporation größer als die Auswaschung, lassen sich diese auch Monate später erkennen. Zusammenfassend kann gesagt werden, dass die Plasmaproteinbindung der GCs zur Verhältnisverschiebung der Konzentrationen im Blut, Speichel und Haar beiträgt. Etwa 50 % des beobachteten Effekts kann der PPB zugeordnet werden. Weitere Quellen sind die unterschiedliche Lipophilie der GCs und die enzymatische Umwandlung, welche im Rahmen der vorliegenden Arbeit jedoch nicht „quantitativ“ betrachtet wurden. Die enzymatische Inaktivierung wurde bis dato als Hauptverantwortliche für die Konzentrationsverschiebung diskutiert. Mit der aktuellen Arbeit wurde dies widerlegt, und die Plasmaproteinbindung als Hauptquelle identifiziert.

Sand flies (Diptera, Phlebotominae) are small biting insects and vectors of Leishmania spp. which cause medically and veterinary important disease - leishmaniasis. During the piercing of the host skin, sand fly females inject saliva to facilitate the blood feeding. The sand fly saliva is composed of many bioactive molecules which were shown to possess anti-inflammatory and anti-haemostatic functions. The saliva affects host's immunity in the bite site and consequently enhances the survival and development of transmitted pathogens. Most of the studies focus on salivary proteins and enzymes of sand flies belonging to Phlebotomus and Lutzomyia genera, while salivary proteins from sand flies of the third genus Sergentomyia were neglected so far. In this thesis we focused on comparison of salivary proteins from two Phlebotomus species, namely Phlebotomus perniciosus and Phlebotomus orientalis, and Sergentomyia schwetzi. These sand fly species differ not only by the ecology and geographical distribution but also by host preferences. Both Phlebotomus species prefer large or medium-size mammals as the bloodmeal source, particularly rabbits, hares and dogs for P. perniciosus and cattle, goats, sheep and humans for P. orientalis. Contrarily, Sergentomyia sand flies are known for preferred feeding on reptiles...

Celiaki (glutenintolerans) är en kronisk, autoimmun sjukdom med diffusa symtom. Vid förtäring av glutenhaltig mat uppstår en allergisk reaktion hos glutenintoleranta individer. Gluten kan inte fullständigt brytas ned av kroppens enzymer, vilket betyder att icke nedbrutna peptidfragment (såsom glutamin) absorberas i tarmslemhinnan. Enzymet transglutaminas katalyserar omvandlingen av glutamin till glutamat. Glutenkänsliga T-celler aktiveras av glutamat att utsöndra proinflammatoriska cytokiner såsom interferon-gamma (IFN-γ) och tumörnekrosfaktor-alfa (TNF-⍺). Syftet med studien var att undersöka om gluten- och gliadinstimulering av celler i helsaliv in vitro kan inducera produktion av IFN-γ och TNF-⍺. Pilotstudier med 2 försökspersoner utfördes, där celler i salivprover stimulerades med enzymatiskt nedbrutet gliadin (<40 mg gliadin), samt PHA (5 µg/mL), PMA (50 ng/mL) och LPS (1 µg/mL) 20 timmar vid 37 ˚C. Cytokinproduktionen i salivproverna kvantifierades med ELISA och uppreglering av IFN-γ och TNF-⍺ undersöktes med RT-qPCR. Efter metodutveckling upprepades stimulering och ELISA med salivprov från 12 försökspersoner (6 individer med och utan celiaki). Immunreaktionen som uppstår hos glutenintoleranta individer in vivo kunde inte återskapas i saliv in vitro med den framtagna metoden. Hos övervägande delen av salivproverna var cytokinproduktionen under detektionsgränsen, 4 pg/mL för IFN-γ och 15,6 pg/mL för TNF-⍺. Det finns risk för att outforskade detaljer eller agens saknades från reaktionskedjan och därmed kunde den förväntade immunreaktionen inte återskapas. En annan felkälla kan vara för låg koncentration av immunceller i saliven. / Celiac disease is a chronic, autoimmune disease that has diffuse symptoms. Upon consuming gluten containing food, an allergic reaction occurs in gluten-sensitive individuals. Gluten cannot be fully digested by human enzymes, which leads to non-digested peptide fragments (such as glutamine) to be absorbed in the gastrointestinal wall. The transglutaminase enzyme catalyzes the conversion of glutamine to glutamate. Glutamate activates gluten-specific T-lymphocytes to produce proinflammatory cytokines e.g., interferon-gamma (IFN-γ) and tumor necrosis factor alpha (TNF-⍺). The aim of this study was to investigate whether stimulation of cells in whole saliva in vitro with gluten and gliadin can induce production of IFN-γ and TNF-⍺. Pilot studies were conducted, where cells in saliva from 2 subjects was stimulated with enzymatically digested gliadin (<40 mg gliadin) together with PHA (5 µg/mL), PMA (50 ng/mL) and LPS (1 µg/mL) for 20 hours at 37 ˚C. The production of cytokines was quantified by ELISA, and the upregulation of IFN-γ and TNF-⍺ was analyzed by RT-qPCR. After method development, the stimulations and ELISA quantifications of the proinflammatory cytokines were repeated in saliva samples from 12 subjects (6 individuals with and without celiac disease). The immune reaction that occurs in people with celiac disease could not be recreated in saliva in vitro with the developed method. In most of the samples the production of cytokines was under the detection range, 4 pg/mL for IFN-γ and 15,6 pg/mL for TNF-⍺. There is risk of unstudied details or agents missing from the reaction chain, and therefore the expected immune reaction could not be recreated. Another source of error could be low concentration of immune cells in saliva.