Orientador: Maria Elvira Pizzigatti Corrêa / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-22T13:37:39Z (GMT). No. of bitstreams: 1 Feio_PatriciadoSocorroQueiroz_D.pdf: 2959247 bytes, checksum: a2ac8e141dbed328b3bf56ecc39c6db1 (MD5) Previous issue date: 2013 / Resumo: Apesar dos avanços, a doença do enxerto contra o hospedeiro (DECH) ainda é principal complicação pós-transplante alogênico de células-tronco hematopoéticas (alo TCTH), que limita sobrevida e induz maior morbidade nos pacientes. A descoberta de proteínas diferentemente presentes na DECH pode ser capaz de tornar o diagnóstico mais precoce e preciso, além de permitir melhor tratamento. Neste contexto, o estudo dos proteomas busca encontrar proteínas que funcionem como biomarcadores e a utilização da saliva para este fim apresenta vantagens por apresentar coleta não invasiva, indolor e de baixo custo. O objetivo deste estudo foi o de avaliar prospectivamente o proteoma salivar em pacientes submetidos ao alo TCTH e comparar a perfil das proteínas em pacientes que desenvolveram a DECH (DECH+) e os que não desenvolveram (DECH-), elaborando um painel de potenciais biomarcadores para esta doença. Foram incluídos neste trabalho 20 pacientes submetidos ao transplante alogênico de células-tronco hematopoéticas na Unidade de Transplante de Medula Óssea do Hospital de Clínicas da Unicamp. Todos os pacientes foram submetidos à avaliação odontológica e adequação do meio bucal quando necessário, além de serem avaliados para grau de mucosite, parâmetros clínicos de hipossalivação e avaliação das lesões orais de DECH. Foram coletadas amostras de saliva não estimulada em 3 períodos: pré-TCTH, no d+8-10 e d+ 80-100. Adicionalmente, foi avaliado o fluxo salivar dos pacientes em todas as coletas. Todas as amostras foram acondicionadas com inibidores de proteases para avaliação proteômica. A análise das amostras foi realizada por espectrometria de massas após digestão com tripsina e separação dos peptídeos por uma coluna reversa de nanocromatografia líquida. Os dados obtidos foram confrontados com o banco International Protein Index (IPI) human utilizando o software Mascot v.2.3.2.0 ® e os arquivos resultantes da busca foram carregados no software Scaffold ®. As proteínas que mostraram variação em razão de 100% (? 0,5 e >2) entre os grupos DECH+ e DECH- no período d+8-10 para d+80-100 foram consideradas significativas. Não houve correlação estatisticamente significativa dos parâmetros clínicos de hipossalivação com o grupo DECH+. Entretanto na avaliação longitudinal dos pacientes, a presença de saliva espessa e viscosa mostrou-se significativa (p = 0,003). Foram identificadas 69 proteínas nos pacientes avaliados, sendo as mais frequentes relacionadas à defesa e imunidade. No presente estudo, dez proteínas mostraram alteração no grupo DECH+, porém 4 estavam presentes em apenas uma amostra com a doença. Portanto, as proteínas Lactoferroxin-B, IGL@ protein, Submaxillary gland androgen-regulated protein 3B, Cystatin-SA, Cystatin-S e Prolactin-inducible protein foram consideradas como potencias biomarcadores para a doença do enxerto contra hospedeiro / Abstract: The graft-versus-host disease (GVHD) remains the major clinical complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT), limiting survival and inducing major morbity. Differently expressed proteins in GVHD have the potential of improving early and accurate diagnosis, also allow a better treatment. Proteome analysis is emerging for the discovery of proteins which function as biomarkers and saliva can be useful for this purpose because offers an easy, inexpensive, safe, and non-invasive approach. The aims of this study were to evaluate salivary proteome in patients underwent allo-HSCT and compare the proteins expressions between patients with and without GVHD and elaborate a panel of possible biomarkers for this condition. Twenty patients underwent allo- HSCT at the Bone Marrow Transplantation Unit - UNICAMP were enrolled in this study. Patients underwent dental evaluation before HSCT and oral environment stabilization were performed when necessary. Also, an evaluation of grade of mucositis, clinical signs of hyposalivation and oral GVHD lesions. Unstimulated saliva samples were collected on 3 different times: pre-HSCT, d+8-10 e d+ 80-100. Additionally, the unstimulated salivary flow rate was measured. Thus, these samples were stored with protease inhibitors for proteomic evaluation. The samples were analyzed by mass spectrometry after protein digestion with trypsin and peptides separated by a Reverse Phase-nanoUltra Perfomance Liquid Chromatography. Protein search was performed using the Mascot v.2.3.2.0 ® engine against International Protein Index (IPI) human Database and the output files of the searches were loaded onto Scaffold software ®. The proteins which showed 100% fold change (? 0,5 e >2) between d+8-10 and d+80-100 were significant. There was no statistically significant correlation of clinical parameters of hyposalivation with GVHD+ group. However in the longitudinal assessment of patients, the presence of thick and viscous saliva was significant (p = 0,003). Sixty nine proteins were identified and the majority was involved in defense and immunity. The results indicated ten proteins differentially expressed in GVHD+ group, but 4 were presented only in one sample with disease. Therefore, Lactoferroxin-B, IGL @ protein, Submaxillary gland androgen-regulated protein 3B, Cystatin SA, Cystatin-S and Prolactin-inducible protein were identified as potential biomarkers for graft-versus-host disease / Doutorado / Patologia / Doutora em Estomatopatologia

O complexo salivar da sanguessuga H. depressa é composto pelas glândulas salivares e probóscide. Análises bidimensionais (2D) mostraram que a maioria das proteínas apresentam pI entre 3.5 à 9.5 e MM entre 10 à 105 kDa. O mapa 2D do complexo salivar apresentou 352 spots totais, sendo 249 exclusivos da saliva (corado por nitrato de prata) e 219 spots totais (corado por Coomassie Blue). As proteínas foram identificadas após sequenciamento tandem MS (proteoma) pela complementariedade às sequências traduzidas do cDNA (transcriptoma). As proteínas mais abundantes foram: antiagregante plaquetário; miohemeritrina e anidrase carbônica. Estas proteínas devem exercer um papel na digestão ou anticoagulação do sangue durante a alimentação. A zimografia também identificou uma protease gelatinolítica (45 kDa). Porém, lefaxin (inibidor de FXa) e hementerina (fibrinogenolítico e inibidor de agregação plaquetária) não foram identificados por estas técnicas biotecnológicas, o que mostra a necessidade de técnicas adicionais para a completa elucidação da constituição desta amostra. / The salivary complex from H. depressa leech is composed of salivary gland and proboscis. Two-dimensional (2D) analysis showed that majority of proteins have pI from 3.5 to 9.5 and MW from 10 to 105 kDa. The 2D gel of salivary complex showed 352 total spots, 249 of them were from saliva (silver stained) and 219 total spots (Coomassie Blue stained). Proteins were identified after tandem MS sequencing (proteome) and complementar analysis of translated sequences from cDNA (transcriptome). The most abundant proteins were: antiplatelet protein; myohemerytrin; carbonic anhydrase. These proteins may have a role in digestion or antihemostatic system during blood feeding. The zymographic assay identified a gelatinolytic protein (45 kDa). But, lefaxin (inhibitor of Fxa) and hementerin (fibrinogenolytic and antiplatelet function) were not identified by these biotechonolgical techniques, showing that additional techniques are necessary for complete knowledge about the composition of this sample.

Travelling wave ion mobility spectrometry (TWIMS) in combination with ultra-high performance liquid chromatography (UHPLC) and mass spectrometry (MS) has been applied successfully for the untargeted, global metabolic profiling of biofluids such as mouse plasma and saliva. Methods based on UHPLC-MS alone and in combination with ion mobility spectrometry (UHPLC-IM-MS) have been developed and validated for the untargeted metabolite profiling of saliva, obtained non-invasively by passive drool. Three separate metabolic profiling studies have been carried out in conjunction with bioinformatics strategies to identify potential metabolomic biomarker ions that are associated with efficacy of rice bran in colorectal cancer, physiological stress and that have the potential for the diagnosis of asthma. The advantages offered by the utility of ion mobility in UHPLC-MS based metabolic profiling studies, including the increased analytical space, mass spectral clean-up of contaminants such as PEG post-UHPLC-IM-MS analysis, enhancement of the selectivity of targeted metabolites as well as the potential for the identification of metabolites by comparison of ion mobility drift times have been highlighted. Ten potential metabolic biomarker ions of asthma have been identified from the moderate asthmatics from untargeted metabolite profiling of saliva by UHPLC-MS. A predictive model based on partial least squares discriminant analysis (PLS-DA) has been constructed using these ten discriminant ions, which demonstrates good predictive capability for moderate asthmatics and controls. Potential metabolic biomarker ions of physiological stress have been identified through untargeted metabolite profiling analysis of saliva samples collected before and after exercise by UHPLC-IM-MS. Valerolactam has been identified as a potential biomarker of physiological stress from saliva by comparison of retention time, ion mobility drift time and MS/MS spectra with a standard of δ-valerolactam.

The life of a preterm infant admitted to a neonatal intensive care unit may be stressful from the moment of birth. Ever since Hans Selye’s initial characterisation of the biological stress response, cortisol has been frequently measured as an indicator of stress responsivity. However, research of the stress response and cortisol in infants, especially those who are preterm and/or ill, has been scarce basically because of methodological issues. The first aim with this thesis was to investigate the acute stress response, as measured by salivary cortisol and behaviour, for preterm infants, healthy infants, and infants at high psychosocial risk in response to certain defined handling procedures. The second aim was to investigate the stress response, as measured by salivary cortisol and psychometric measures, for parents present during the handling procedure of their infants. The intention was to perform all investigations in an as naturally occurring situation as possible, which means that the studied procedures would have been performed irrespectively of the research. The present thesis includes six original articles. The results of the first study demonstrate that it is feasible to collect sufficient amounts of saliva and to analyse salivary cortisol in neonates using the presented method of collection and analysis. The second study shows that preterm infants, usually cared for in incubators, show no signs of discomfort and have variable cortisol responses during skin-to-skin care with their mothers. The mothers, however, experience stress and low control before their first skin-to-skin care with their preterm infant and do not relax completely until after the session. In the third study we found that preterm infants have higher baseline salivary cortisol as compared to healthy full-term infants. Moreover, preterm infants have higher and sustained pain response during a nappy change as compared to healthy full-term infants. The results of the fourth study shows that infants younger than three months, living in psychosocial high-risk families, have increased cortisol responses during a nappy change, performed by the mother. However, support with the aim of improving mother-infant interaction, dampens the stress response. The results of the fifth study show that oral sweet-tasting solution in combination with a pacifier dampen the levels of the stress hormone cortisol in three months old infants during routine immunisation. Moreover, parents experience more self-rated emotional stress before immunisation if it is their first child who is being immunised. The sixth paper shows that the material used for saliva collection (cotton buds with wooden or plastic sticks) is of importance when saliva is collected but for practical reasons not centrifuged within 24 hours prior to cortisol analyse. The present thesis shows that it is practically feasible to collect saliva and to analyse the stress hormone cortisol in infants. The interpretation of infants’ and parents’ salivary cortisol responses to different handling procedures are discussed in relation to shortand long-term consequences, neonatal intensive care, preterm birth, attachment, mood, and pain.

Despite the abundance of research conducted into the effects of exercise on mucosal immunity the results remain controversial. Much of the inconsistencies arise from the exercise protocols, the participants studied and their nutritional status, as well as methodological and analytical differences. The purpose of this thesis was to examine the influence of some of these factors, and to investigate potential means of enhancing the mucosal immune response to exercise. In study 1 (Chapter 3) it was shown that a fed or fasted state 2 h prior to exercise had no effect on the s-IgA concentration or secretion rate during prolonged exercise. However, when participants were fed during exercise (Chapter 4), the secretion rate of salivary antimicrobial proteins lysozyme and a-amylase increased, but sIgA remained unchanged. These changes were likely due to the activation of mechanical and gustatory receptors leading to a reflex stimulation of protein secretion via the autonomic nerves, rather than changes in stress hOnliones, since cortisol did not change significantly during exercise. Study 3 (Chapter 5) extended these findings where it was demonstrated that chewing flavoured gum during exercise enhanced lysozyme and a-amylase secretion but resulted in a small reduction in s-IgA secretion rate. Salivary antimicrobial proteins are affected by the exercise intensity since both s-IgA and lysozyme secretion rate increased post -exercise following an incremental test to exhaustion, but not after exercise at 50% Y02max. Moreover, lysozyme secretion rate was also elevated following exercise at 75% Y02mru<, whereas s-IgA remained unchanged. These effects are thought to be mediated by increased sympathetic nervous system activity reflected by the concomitant increases in (lamylase and chromogranin A, rather than the hypothalamic-pituitary-adrenal axis. Resting mucosal immunity exhibits significant gender differences. In study 1 (Chapter 3) s-IgA concentration, secretion rate and osmolality were found to be lower in females than in males at rest. In addition, saliva flow rate was found to be lower in females compared with males in study 5 (Chapter 7). However, these differences did not appear to influence the salivary responses to acute exercise or exercise training. Chronic exercise training in elite male and female swimmers resulted in lower levels of s-IgA secretion rate following periods of intense training prior to competition compared with post-competition (Chapter 7), but these levels were not directly associated with reported episodes of respiratory illness.

Protein identification and quantitation using mass spectrometry has evolved as the dominant technique for studying the protein complement of a system: cell, tissue or organism. The proteomics of body fluids is a very active research area as there is great potential for protein biomarker discovery; application of such technologies would revolutionise medical practice and treatment. Saliva, through its non intrusive nature of sampling, is an ideal body fluid for disease diagnosis, screening and monitoring. Gingivitis is a gum disease with symptoms including bleeding, swollen, and receding gums. After dental decay, gingivitis is estimated to be the most common disease worldwide, and around 40% of the population in the US are reported to have gingivitis. The end point goal of this project was to identify salivary biomarkers for gingivitis. This dissertation presents an investigation of: 1) the salivary proteome; 2) developments and applications of a mass spectrometry kinase assay; and 3) salivary biomarkers for gingivitis using proteomics and kinase activities. The soluble portion of the human salivary proteome (saliva supernatant) has been studied by several research groups but very few proteomic studies have been performed on the insoluble, cellular and bacterial portion of saliva. Presented here, is the first global proteomics study performed on the saliva residue and supernatant from the same test subject. A total of 834 and 1426 proteins were identified in the saliva supernatant and residue, respectively. A global analysis of protein complexes in saliva was also performed and is the first study, to date, of such an analysis. KAYAK (‘Kinase ActivitY Assay for Kinome analysis’) was further developed for its application on a number of cell types, tissue types, and a variety of organisms. Proof of concept work for in-gel kinase activity/kinase abundance correlation profiling using blue native gels was performed, and experiments using anion exchange chromatographic kinase activity/kinase abundance correlation profiling were performed to identify kinase-substrate pairs. KAYAK applications included the analysis of kinase activities in Saccharomyces cervisiae, Drosophila, mouse, and human saliva in which significant kinase activity was detected in the saliva supernatant, a novel finding. Finally, gingivitis was induced in patients, and the saliva samples were analysed using proteomics and kinase activity profiling. Although this work is ongoing, preliminary data indicate that there are increases in various inflammatory proteins, certain bacteria and also in the activity of particular kinases as a result of the induction of gingivitis. The overall study provided insights into the salivary proteome for both the human and bacterial complement, as well as discovering the presence of significant kinase activity in saliva. In the induced gingivitis study, almost half of all the proteins identified in the residue were from bacteria (1274 bacterial proteins, 198 species identified) and there may be more potential for biomarker discovery for certain diseases in the saliva residue than in the supernatant. A very large overlap was observed between the human proteins in the saliva supernatant and residue, indicating that many of the salivary proteins originate from lysed cells. The origin of the kinase activity in the saliva supernatant is not known but is also proposed to originate predominantly from lysed cells. A range of novel KAYAK applications have been investigated, demonstrating that KAYAK has a wide variety of future uses ranging from target compound evaluation in Pharmaceutical companies to patient testing in the clinic.

Con el objetivo de determinar la efectividad antibacteriana in vitro de una infusión a base de Camelia sinensis y Minthostachys mollis sobre flora salival mixta, se recolectó saliva no estimulada de 30 estudiantes universitarios y se sembró en el medio Agar Tripticasa soya. Utilizándose el método de difusión por discos para las soluciones de té verde, muña, té verde y muña, y los controles positivo (Clorhexidina) y negativo (Agua destilada), las placas se incubaron a 37° C / 24 horas. El análisis estadístico determinó la efectividad antibacteriana de las infusiones a base de té verde, y té verde y muña, no encontrando efectividad en la infusión a base de muña, y que existían diferencias significativas entre las medias de las muestras. Así mismo, la infusión a base de té verde resultó ser similar en cuanto a su efectividad antibacteriana con respecto a la clorhexidina. De los resultados obtenidos se concluye que se ha evidenciado la efectividad antibacteriana de una infusión a base de té verde y muña sobre la flora salival mixta, sin embargo, se observó una efectividad antibacteriana menor con respecto a la infusión a base de té verde y la clorhexidina. Debido a los resultados del presente estudio que demuestran la efectividad del té verde similar a la clorhexidina, es necesario la continuación de estudios in vivo para comprobar dicho hallazgo y promover su uso como enjuagatorio bucal, así como estudios de asociación de infusión de té verde con aceite esencial de muña o con infusión de muña a mayor concentración.

Se realizó investigación de tipo casi-experimental cruzado comparativo en el Puericultorio Pérez Araníbar, con el objetivo de determinar el pH salival sometido a cuatro diferentes situaciones: dieta cariogénica y no cariogénica con y sin cepillado previo. Se trabajó con una muestra de 30 niños agrupados según sexo (niños y niñas) y según grados de afectación por caries dental: 0, no presenta; 1, 1 a 4 lesiones; 2, más de 4 lesiones. Se recolectó saliva total con el método Spitting, tomándose cuatro muestras: 5 minutos antes, 10, 20 y 40 minutos después del desayuno. / -- The research was realized in Pérez Aranibar Puericulture Institution. The purpose of this research was to determinate the salivary pH when it is submitted to four different situations: cariogenic and non cariogenic diet with and without previous toot brushing. The study included 30 children who were grouped according to sex (boy and girl) and the different decay affectation grade: 0, doesn’t show; 1, 1 to 4 lesions; 2, more than 4 lesions. The total saliva was gathered using the Spitting method and four samples of saliva: 5 minutes before, 10, 20 and 40 minutes after breakfast were taken.

El objetivo de la presente investigación fue determinar la relación entre las concentraciones de calcio y fósforo en saliva estimulada con la presencia y ausencia de cálculo supragingival. Se evaluaron muestras de saliva total estimulada de personas sanas, que acudieron al “Consultorio Dental de Tropa de la División de Estomatología del Hospital Militar Central”, Lima-Perú; 25 presentaban cálculo supragingival y 25 no lo presentaban, sus edades estuvieron entre los 17 y 21 años, incluidos hombres y mujeres. Se realizó un análisis espectrofotométrico a las muestras de saliva para determinar las concentraciones de calcio y fósforo. Se observó que existió una diferencia significativa para el elemento fósforo (p=0,047) al comparar los 2 grupos de estudio, pero no se observó lo mismo en el caso del calcio (p=0,094). Las concentraciones de calcio y fósforo encontradas en el presente estudio fueron similares a las concentraciones mínimas encontradas en estudios de otros países. Se demuestra así, que la participación del ión fósforo en la formación del cálculo supragingival es importante y que por lo tanto interviene en el estado de salud de la cavidad oral. / The objective of the present investigation was to determine the relation between the concentrations of calcium and phosphorus in saliva stimulated with the presence and absence of supragingival calculus. Samples of stimulated total saliva of healthy people were evaluated, who went to the “Consultorio Dental de Tropa del Hospital Militar Central”, Lima-Perú; 25 presented supragingival calculus and 25 did not present it, their ages were between the 17 and 21 years, including men and women. A spectroscopy analysis to the saliva samples was made to determine the concentrations of calcium and phosphorus. It was observed that a significant difference for the element existed phosphorus (p=0,047) when comparing the 2 training groups, but was not observed the same in the case of calcium (p=0,094). The concentrations of found calcium and phosphorus in the present study were similar to the found minimum concentrations in studies of other countries. One demonstrates thus, that the participation of the ion phosphorus in the formation of the supragingival calculus is important and that therefore it takes part in the state of health of the oral cavity.

La fitoterapia es la terapia basada en plantas, alimentos y elementos nutritivos. Se encuentra entre las más antiguas. Esta terapia utiliza esencias puras de plantas para tratar diversos problemas, así dermatológicos, alérgicos, digestivos, ginecológicos, etc. Disminuyendo los efectos secundarios y haciendo más efectivos los tratamientos. La fitoterapia a través de los extractos naturales y sus destilaciones artificiales constituye la base de la medicina moderna y de la cosmética actual. Se sabe que multitud de pueblos descubrieron ya en tiempos remotos, que algunas plantas eran buenas para comer y alimentarse y otras se caracterizaban por tener propiedades curativas. Con toda certeza, la búsqueda de algún remedio fue la génesis del uso de las plantas para su propio beneficio, ya fuera fruto del deseo de sanar o por cuestión mágico-religiosa, siendo simplemente en la mayoría de los casos con motivo de la búsqueda de nuevos alimentos. Los egipcios, desarrollaron la utilización de las plantas medicinales de forma sistemática y estructurada, conociéndose más de 700 fórmulas en las que aparecen plantas curativas, destacando el impreso más importante, el Papiro de Ebers, 1700 A.C., pudiendo atribuirle un origen anterior en Asia. En China se supone que ya era utilizada en el 5000 A.C., destacando el libro de Pen Tsao que recoge el estudio de más de 300 plantas. En la India se menciona la utilización de las plantas medicinales en Rig Veda, uno de los libros sagrados del brahmanismo. El Ayurveda o como se conoce el uso de las plantas medicinales en la India, hace referencias escritas al año 800 A.C., describiendo unas 800 especies. El conocimiento de las plantas medicinales se extendió desde el Antiguo Egipto y Mesopotamia hacia los países mediterráneos, hasta Grecia y luego por toda Europa para llegar 2000 años más tarde al Nuevo Mundo. El conocimiento de las plantas medicinales, ya sea a través de la magia, religión, necesidad o casualidad, o a veces como consecuencia del ensayo-error ha permitido obtener un conocimiento de las plantas medicinales entre las diferentes culturas que constituyen la base de la medicina moderna, sabiduría que nos corresponde a todos conocer y salvaguardar como parte de nuestro patrimonio. En la actualidad ha resurgido el interés público y científico en el desarrollo de la medicina natural tradicional con el impulso de nuevas tecnologías que descubren cada día nuevas propiedades y aplicaciones a los diferentes principios activos de las plantas, subrayando el carácter dinámico y de servicio en beneficio de la humanidad en el estudio de las plantas medicinales. La odontología no escapa de estos antecedentes históricos, que nos ofrecen una alternativa a tomar en cuenta, y a la vez nos estimula para asumir con responsabilidad nuevos retos que nos llevan a proponer recursos novedosos para el control y tratamiento de las enfermedades estomatológicas con mayor prevalencia en nuestra población.