Дисертації з теми "Salivary markers"

The aim of this review is to discuss current findings regarding the roles of miRNAs in periodontal diseases and the potential use of saliva as a diagnostic medium for corresponding miRNA investigations. For periodontal disease, investigations have been restricted to tissue samples and five miRNAs, that is, miR-142-3p, miR-146a, miR-155, miR-203, and miR-223, were repeatedly validated in vivo and in vitro by different validation methods. Particularly noticeable are the small sample sizes, different internal controls, and different case definitions of periodontitis in in vivo studies. Beside of that, the validated miRNAs are associated with inflammation and therefore with various diseases. Furthermore, several studies successfully explored the use of salivary miRNA species for the diagnosis of oral cancer. Different cancer types were investigated and heterogeneous methodology was used; moreover, no overlap of resultswas found. In conclusion, fivemiRNAs have consistently been reported for periodontitis; however, their disease specificity, detectability, and expression in saliva and their importance as noninvasive markers are questionable. In principle, a salivary miRNA diagnostic method seems feasible.However, standardized criteria and protocols for preanalytics, measurements, and analysis should be established to obtain comparable results across different studies.

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Introdução: Anualmente milhões de mulheres em todo o mundo são rastreadas para câncer de mama, sendo que algumas delas serão submetidas a procedimentos adicionais, entre eles a biópsia mamária. A biópsia de fragmento (core biopsy) guiada por ultrassonografia é um método minimamente invasivo para o diagnóstico de lesões mamárias suspeitas, e possui inúmeras vantagens quando comparada às biópsias cirúrgicas. A core biopsy é um procedimento ambulatorial no qual raramente é necessário o uso de medicações analgésicas após o procedimento. No entanto, qualquer procedimento invasivo, por menor que seja, vem acompanhado de experiências de ansiedade, dor e medo do desconhecido. A Medicina Anti-Estresse entende que os pensamentos, sentimentos, níveis de ansiedade e capacidade de lidar com eventos estressores podem afetar a saúde e a percepção de saúde. Programas de Medicina Anti Estresse reduzem o estresse e auxiliam os praticantes a terem atitudes positivas em seu cotidiano. O objetivo deste estudo controlado e randomizado foi avaliar o impacto das intervenções baseadas em Mindfulness (MBIs) em parâmetros quantitativos e qualitativos de estresse em pacientes submetidas a biópsias de mama guiadas por imagem. Métodos: Após assinarem o consentimento informado, 82 mulheres com indicação de biópsia de mama guiada por imagem foram randomizadas em dois grupos: grupo MBI ou grupo controle de cuidados padrão. Na semana anterior à biópsia, minutos antes na sala de espera e durante o procedimento, o grupo MBI foi exposto a meditações guiadas por áudio e músicas relaxantes e o grupo controle recebeu suporte da equipe de biópsia por meio de diálogo. As participantes preencheram questionários que avaliam depressão, ansiedade e estresse (DASS-21), dados demográficos e histórico médico, além de terem avaliado sua experiência de dor por meio de uma Escala Visual Analógica de Dor (EVA) e tiveram sua pressão arterial sistólica e diastólica, temperatura inicial e final, frequência cardíaca, saturação de oxigênio e cortisol salivar medidos. Resultados: A participação no grupo de intervenção foi associada a níveis reduzidos de estresse, pressão arterial, frequência cardíaca e saturação de oxigênio em comparação com a participação no grupo de cuidados padrão (valores de P < 0,05). Não houve diferença em relação aos níveis salivares de cortisol, temperatura periférica e percepção de dor entre os dois grupos estudados. Conclusão: Os resultados indicam que intervenções extremamente breves em Mindfuness são viáveis e aceitáveis, sugerindo que programas baseados em atenção plena podem ser benéficos para reduzir o desconforto em situações de estresse agudo.
Introduction: Each year worldwide millions of women are screened for breast cancer and some of them will undergo additional investigations, including breast biopsy. Ultrasound guided core needle biopsy is a minimally invasive method for the diagnosis of suspected mammary lesions and has many advantages when compared to surgical biopsies. Core biopsy is an outpatient procedure and analgesic medication is rarely necessary after tissue removal. Nevertheless, any invasive procedure, however small it may be, is accompanied by experiences of anxiety, pain and fear of the unknown. Anti Stress Medicine recognizes that thoughts, feelings, levels of anxiety, and ability to cope with stressful events can affect health and health perception. Anti-Stress Medicine programs reduce stress and help practitioners to have positive attitudes in their daily lives. The purpose of this randomized controlled trial was to evaluate the impact of Mindfulness-based interventions (MBIs) on quantitative and qualitative stress parameters on patients undergoing imaging-guided breast biopsies. Methods: After giving informed consent, 82 women that needed percutaneous imaging-guided breast biopsy were randomized into two groups: MBI group or standard care control group. One week before the biopsy procedure, minutes prior to the biopsy procedure on the waiting room and during the biopsy procedure, the MBI group was exposed to audio guided meditations and relaxing music and the standard care control group received supportive dialogue from the biopsy team. Participants completed questionnaires measuring depression, anxiety and stress (DASS-21), demographics, and medical history, evaluated their pain experience through a Visual Analogue Scale for Pain (VAS) and had their systolic and diastolic blood pressure, initial and final temperate, heart rate, oxygen saturation and cortisol salivary measured. Results: Participation in the mindfulness intervention group was associated with reduced levels of perceived stress, blood pressure, heart rate and oxygen saturation compared to participation in the standard care group (P values < 0.05). No difference was observed regarding salivary cortisol levels, peripheral temperature and pain perception between the two studied groups. Conclusion: Results indicate that an extremely brief mindfulness intervention is a feasible and acceptable intervention, suggesting that Mindfulness-based programs may be beneficial to reduce discomfort in acutely stressful settings.

Orientador: Agnaldo Lopes da Silva Filho
Resumo: Introdução: Anualmente milhões de mulheres em todo o mundo são rastreadas para câncer de mama, sendo que algumas delas serão submetidas a procedimentos adicionais, entre eles a biópsia mamária. A biópsia de fragmento (core biopsy) guiada por ultrassonografia é um método minimamente invasivo para o diagnóstico de lesões mamárias suspeitas, e possui inúmeras vantagens quando comparada às biópsias cirúrgicas. A core biopsy é um procedimento ambulatorial no qual raramente é necessário o uso de medicações analgésicas após o procedimento. No entanto, qualquer procedimento invasivo, por menor que seja, vem acompanhado de experiências de ansiedade, dor e medo do desconhecido. A Medicina Anti-Estresse entende que os pensamentos, sentimentos, níveis de ansiedade e capacidade de lidar com eventos estressores podem afetar a saúde e a percepção de saúde. Programas de Medicina Anti Estresse reduzem o estresse e auxiliam os praticantes a terem atitudes positivas em seu cotidiano. O objetivo deste estudo controlado e randomizado foi avaliar o impacto das intervenções baseadas em Mindfulness (MBIs) em parâmetros quantitativos e qualitativos de estresse em pacientes submetidas a biópsias de mama guiadas por imagem. Métodos: Após assinarem o consentimento informado, 82 mulheres com indicação de biópsia de mama guiada por imagem foram randomizadas em dois grupos: grupo MBI ou grupo controle de cuidados padrão. Na semana anterior à biópsia, minutos antes na sala de espera e durante o procedimento... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Introduction: Each year worldwide millions of women are screened for breast cancer and some of them will undergo additional investigations, including breast biopsy. Ultrasound guided core needle biopsy is a minimally invasive method for the diagnosis of suspected mammary lesions and has many advantages when compared to surgical biopsies. Core biopsy is an outpatient procedure and analgesic medication is rarely necessary after tissue removal. Nevertheless, any invasive procedure, however small it may be, is accompanied by experiences of anxiety, pain and fear of the unknown. Anti Stress Medicine recognizes that thoughts, feelings, levels of anxiety, and ability to cope with stressful events can affect health and health perception. Anti-Stress Medicine programs reduce stress and help practitioners to have positive attitudes in their daily lives. The purpose of this randomized controlled trial was to evaluate the impact of Mindfulness-based interventions (MBIs) on quantitative and qualitative stress parameters on patients undergoing imaging-guided breast biopsies. Methods: After giving informed consent, 82 women that needed percutaneous imaging-guided breast biopsy were randomized into two groups: MBI group or standard care control group. One week before the biopsy procedure, minutes prior to the biopsy procedure on the waiting room and during the biopsy procedure, the MBI group was exposed to audio guided meditations and relaxing music and the standard care control group receiv... (Complete abstract click electronic access below)
Doutor

Syfte: Att utreda sambandet mellan den lösliga formen av urokinas-receptorn (suPAR) i saliv hos patienter med artrit i käkleden (A-TMJ) och friska kontroller, för att skapa en grund för vidare forskning av suPAR som prediktor för inflammationsgraden i käkleden hos patienter med A-TMJ.Material och metod: En fall-kontrollstudie utfördes med 6 kontroller (medelåldern 31±11år) och 5 patienter med A-TMJ (medelåldern 24±5år). Undersökningen bestod av salivprov, registrering av blödning vid sondering (BoP), blodprovstagning, och undersökning av tuggsystemet där antalet smärtsamma käkledsrörelser (PM) mättes. Sist samlades käkledvätska in. Halten suPAR analyserades i saliv, plasma och käkledsvätska. Resultat: En signifikant skillnad mellan suPAR i saliv kunde ej påvisas (A-TMJ 4,4±3,91ng/ml, kontroller 4,96±4,80ng/ml), emellertid hade patienter en signifikant högre halt av suPAR i plasma (A-TMJ 2,71±0,62ng/ml, kontroller: 1,86±0,35ng/ml, P=0,017). Halten av suPAR i käkledsvätska mättes till 1,57±1,50ng/ml hos patienter men kunde inte detekteras hos kontroller. BoP mättes till 16±9% hos patienter och 14±7% hos kontroller, och median(IQR) för PM var 3(1) i höger käkled och 0(3) i vänster käkled hos patienter. Slutsatser: (i) Ingen slutsats kan dras gällande sambandet mellan suPAR i saliv och A-TMJ, men (ii) patienter med A-TMJ har till viss mån en högre smärta i käkleden vid käkledsrörelse medan deras koncentration av suPAR i plasma är högre jämfört med friska kontroller. Det verkar som (iii) BoP skulle kunna vara kopplat till suPAR i saliv. Resultat från denna studie bör tolkas med försiktighet på grund av litet stickprov, fortsatt forskning behövs för att klargöra sambandet mellan suPAR i saliv och A-TMJ.
Aims: To investigate the levels of soluble urokinase plasminogen activator receptor (suPAR) in saliva between patients with arthritis in the temporomandibular joint (A-TMJ) and healthy controls to create a foundation for further research of the potential predictive value of suPAR in patients with A-TMJ.Materials and method: A case- control study was conducted, 6 controls (mean age 31±11years) and 5 patients with A-TMJ (mean age 24±5years) enrolled in the study. Saliva, blood, synovial fluid (SF) were sampled, and the masticatory system was examined according to DC/TMD, and bleeding on probing (BoP) was assessed, as was painful mandibular movement (PM). The level of suPAR was analyzed in saliva, plasma and SF.Results: Level of salivary suPAR did not differ significantly between A-TMJ patients and healthy controls (P > 0.05). Patients had a significantly higher level of suPAR in plasma than controls (A-TMJ 2.71±0.62ng/mL, controls: 1.86±0.35ng/mL, P=0.017). suPAR level in SF was measured to 1.57±1.50ng/mL in A-TMJ patients and not detected in controls. BoP was 16±9% in patients and 14±7% in controls, and median(IQR) of PM was 3(1) in the right TMJ and 0(3) in the left in patients.Conclusions: (i) No conclusion can be drawn regarding suPAR in saliva and A-TMJ, but (ii) to some degree A-TMJ patients have higher PM meanwhile their plasma concentration of suPAR is higher than controls. A trend that (iii) higher BoP is connected with higher suPAR in saliva could be distinguished. Results must be interpreted with caution due to small study sample, more research is required to further elucidate the association between suPAR in saliva and A-TMJ.

The thesis examines acute and chronic effects of physical training or competition on the kinetics of selected clinical and psychological markers, together with associations between some of these markers and other variables in three exercising groups.

Attempts have been made to account for the increased propensity of upper respiratory tract infections (URTI) in regularly training individuals, by focusing on the effect of exercise on the primary mucosal immunoglobulin secretory immunoglobulin A (sIgA). However, the pathophysiological significance of observed changes in sIgA levels remains unclear, as indeed does the existence of increased URTI in athletes. The aim of this work was to examine the use of sIgA as a marker of exercise-induced immunomodulation and its possible role in protection against URTI. Before these questions could be addressed the optimum method for saliva collection, and the ideal terms for expression of sIgA levels were investigated. Experimental data advocate the use of the passive dribbling method above the use of salivettes. Salivettes were found to result in an underestimation of both saliva flow rate and protein concentrations as a consequence of limited absorption (3 ± 1ml) and variable retention of the sample (49.1 ± 24.9%). Secretory IgA levels are commonly expressed in terms of secretion rate which is the product of saliva flow rate and sIgA concentration. Examination of the relative contribution of these two factors to secretion rate revealed that exercise-induced changes in saliva flow rate (-51 %) matched the changes in secretion rate (-51 %), whereas changes in sIgA concentrations (+4%) did not. It was concluded that changes in saliva flow rates have an important role with the occurrence of symptoms associated with URTI whether induced by infective or inflammatory factors. Epidemiological data from others on the incidence of symptoms associated with URTI in marathon runners have provided evidence on the incidence of URTI in athletes. However here, reported symptoms associated with URTI were most common during the race suggesting that an infective agent was not involved. Examination of the effect of marathon running revealed a non-significant decrease in saliva flow rate (-27.7 ± 15.8%). A final study investigated the effect of increased ambient temperature, and the possibility of fluid replacement as a intervention strategy against exercise-induced decreases in saliva flow rate. This study revealed that exercise reduced saliva flow rate exercise in the heat exacerbated this and that fluid replacement tempered the exercise-induced decrease. Changes in saliva flow rate were found to be associated with changes in plasma volume. The overall conclusions of this thesis are that innate defence mechanisms such as saliva have a role to play in conferring defence against potential pathogens, and therefore warrant further investigation. It appears from data presented in this thesis that saliva flow rate is affected by exercise, perhaps to a greater extent than sIgA concentration. Changes in saliva flow rate with exercise may have a role to play in the purported increased incidence of URTI reported by athletes, and fluid replacement may provide an effective strategy against this exercise-induced decrease.

Les athlètes de haut-niveau français, spécialistes des sprints longs en athlétisme, rencontrent des difficultés à maintenir des performances optimales lors des compétitions internationales. Dans ce contexte, l'objectif général de ce travail de thèse a été, à travers une approche holistique, de chercher àoptimiser la réitération d'exercices de haute-intensité et d'améliorer la récupération des athlètes lors des compétitions internationales. Pour ce faire, deux axes relatifs au stress ont été explorés : tout d'abord, nous avons analysé l'impact du stress psychophysiologique en compétition sur la régulation du système nerveux autonome et du système nerveux neuro-endocrinien, ainsi que l'évaluation de stratégies innovantes de gestion du stress telles que la cohérence cardiaque pour améliorer la régulation neuroendocrinienne et la qualité du sommeil des athlètes au cours des compétitions. Par ailleurs, aucours des exercices de haut-intensité, la production énergétique entraîne d'importantes perturbations métaboliques, telles qu'une acidose métabolique marquée (avec un pH sanguin inférieur à 7, relevé à l'issue des épreuves de 400 m), pouvant constituer un frein à la performance. Pour y remédier, nousavons appréhendé un second axe de recherche sur la régulation de l'équilibre acido-basique au cours de l'exercice et la récupération, à travers la mise en place de stratégies nutritionnelles et hydriques, incluant une alimentation spécifique et une hydratation à base d'eau riche en bicarbonates.Les premières études ont révélé des perturbations psychophysiologiques liées au stress compétitif, avec une diminution significative de l'activité du système parasympathique et une augmentation des biomarqueurs salivaires du stress (cortisol et alpha-amylase). Ces perturbations, observées lors desphases qualificatives et en finales, sont corrélées à une anxiété accrue associée à une capacité de récupération réduite des athlètes (études n°1 et n°2). Une variabilité interindividuelle a été constatée, certains athlètes se montrant particulièrement sensibles au stress compétitif, ce qui souligne l'importanced'approches individualisées pour mieux gérer ces réponses. Par ailleurs, l'introduction de la cohérence cardiaque comme méthode de gestion du stress a permis d'améliorer la régulation du système nerveux autonome, de réduire l'inflammation (IL1-β) et de favoriser une meilleure qualité du sommeil, essentielle pour récupérer entre les épreuves. Les athlètes pratiquant cette technique ont montré une meilleure régulation hormonale et inflammatoire, même si aucun impact direct sur les performances en compétition n'a été observé (étude n°3). Puis, les études suivantes de ce travail de thèse, relatives à la régulation du stress métabolique, ont démontré que la consommation d'eau riche en bicarbonates, associée à un régime alimentaire alcalinisant (à la différence d'une régime acidifiant), augmente significativement le pH sanguin et urinaire des athlètes, améliore la clairance du lactate après desexercices répétés et optimise la capacité tampon (études n°4 et n°5). Ces ajustements métaboliques ont permis aux athlètes de maintenir ou d'améliorer leurs performances, notamment lors du troisième jour de compétition à haute-intensité (études n°5).En conclusion, les résultats de ce travail de thèse soulignent l'importance d'intégrer des stratégies combinant la gestion du stress précompétitif, notamment à travers la pratique de la cohérence cardiaque, ainsi que d'avoir une approche nutritionnelle et hydrique adaptée au moment des compétitions internationales. Ces stratégies favorisent en effet une meilleure régulation du système nerveux autonome et du système nerveux neuro-endocrinien, améliorent la qualité du sommeil, et contribuent à une gestion optimisée de l'acidose métabolique induite au cours de la compétition afin d'améliorer à la fois les performances et la récupération des athlètes de haut niveau
French elite athletes specializing in long sprints in track and field face challenges in maintaining optimal performance during international competitions. In this context, the overarching goal of this doctoral research was to adopt a holistic approach to optimize the repetition of high-intensity exercises andenhance athlete recovery during international events. To achieve this, two aspects related to stress were explored: first, the study examined the impact of psychophysiological stress during competition on the regulation of the autonomic and neuroendocrine nervous systems, as well as the evaluation of innovative stress management strategies such as cardiac coherence to improve neuroendocrine regulation and sleep quality during competitions.Additionally, during high-intensity exercises, energy production induces significant metabolic disruptions, such as marked metabolic acidosis (with blood pH below 7 observed after 400 m events), which may hinder performance. To address this, nutritional and hydration strategies, including specific diets and hydration with bicarbonate-rich water, were tested to better regulate metabolic stress.Initial studies revealed psychophysiological disruptions related to competitive stress, with a significant decrease in parasympathetic activity and increased salivary stress biomarkers (cortisol and alpha-amylase). These disruptions, observed during both qualifying phases and finals, correlated withheightened anxiety and reduced recovery capacity (Studies 1 and 2). Interindividual variability highlighted some athletes' heightened sensitivity to competitive stress, emphasizing the need for individualized approaches to better manage these responses.The introduction of coherente breathing as a stress management technique improved autonomic nervous system regulation, reduced inflammation (IL-1β), and enhanced sleep quality, essential for recovery between events. Athletes practicing this technique demonstrated improved hormonal and inflammatory regulation, although no direct impact on competition performance was observed (Study 3).Subsequent studies addressing metabolic stress regulation demonstrated that consuming bicarbonate-rich water combined with an alkalizing diet (as opposed to an acidifying diet) significantly increased blood and urinary pH, improved lactate clearance after repeated exercises, and optimized bufferingcapacity (Studies 4 and 5). These metabolic adjustments enabled athletes to maintain or improve performance, particularly on the third day of high-intensity competition (Studies 5).In conclusion, this research highlights the importance of integrating strategies that combine pre-competitive stress management, particularly through coherent breathing, with tailored nutritional and hydration approaches during international competitions. These strategies promote better regulation ofthe autonomic and neuroendocrine nervous systems, enhance sleep quality, and help manage competition-induced metabolic acidosis. Together, these approaches can improve both performance and recovery for high-level athletes in demanding competitive contexts

Aim: The aim of this thesis was to investigate the use of salivary Epstein-Barr virus (EBV) DNA as a marker of in vivo immunity in response to training and nutritional intervention. Methods: Initially, an assay for the detection of EBV DNA in saliva was developed which was subsequently used to detect the concentration of EBV DNA in samples collected in subsequent studies. The potential role of EBV as a predictor of URTI in response to endurance exercise are presented, along with the outcome of nutritional interventions, with chapters investigating the effects of supplementation with carbohydrate, and Chlorella pyrenoidosa. Finally, data from all four of these chapters were consolidated, and the role of salivary EBV DNA as a marker of in vivo immunity investigated. Outcome: The main finding from this thesis is that salivary EBV DNA does not appear to be a useful marker of in vivo immunity based on the present data. EBV concentration was not a predictor of URTI, nor was there a relationship between EBV concentration and SIgA concentration or secretion rate, or the absolute change or percentage change in EBV from pre-post exercise and the absolute and percentage change in SIgA concentration or secretion rate.

INTRODUÇÃO. A proteína Fator Inibitório da Migração de Macrófagos (MIF) é frequentemente observada com expressão elevada em tecidos tumorais quando comparados aos tecidos equivalentes normais e estudos têm sugerido seu papel como marcador prognóstico de neoplasias malignas, incluindo carcinomas hepatocelular, de ovário, de esôfago e também de cabeça e pescoço. Adicionalmente, alguns de seus mecanismos de ação já demonstrados, como a indução da proliferação e migração celular permitem implicar essa expressão diferencial no desenvolvimento tumoral e, consequentemente, no prognóstico das neoplasias malignas. OBJETIVO. Esse estudo objetivou avaliar o papel diagnóstico e prognóstico da proteína MIF em carcinoma epidermóide da cavidade bucal. METODOLOGIA. O estudo foi composto por 50 pacientes com carcinoma epidermoide da cavidade bucal coletados prospectivamente e 57 casos retrospectivos admitidos nos Serviços de Cirurgia de Cabeça e Pescoço do Hospital Heliópolis e da Faculdade de Medicina da Fundação ABC. As análises foram feitas por meio de imunoistoquímica dos tecidos tumorais e margens epiteliais normais e de ELISA das amostras de soro e saliva, coletadas pré e pós-tratamento cirúrgico, dos pacientes participantes. Os resultados foram correlacionados aos achados clínicos e histopatológicos. RESULTADOS. A expressão da proteína MIF e seu receptor CD74 mostrou-se elevada em tecido tumoral quando comparado ao tecido epitelial livre de neoplasia (p < 0,0001). Associação entre a alta expressão da MIF no tumor e infiltração vascular linfática foi observada (p=0,005) e alta expressão da MIF no epitélio livre de tumor apresentou correlação marginalmente significativa com ocorrência de segundo tumor primário (p=0,072). A expressão positiva do receptor CD74 não apresentou associação com variáveis clínicas ou histopatológicas. A concentração sorológica da proteína MIF apresentou associação inversa com metástase linfonodal (p=0,018) e estádios patológicos avançados (p=0,040) e foi significativamente reduzida após a ressecção do tumor (p=0,001). A concentração da MIF na saliva não apresentou redução significativa após o tratamento cirúrgico, mas foi associada aos estágios pT3 e pT4 (p=0,001) e a estádios patológicos avançados (p=0,032). CONCLUSÕES. A redução significante da concentração da MIF observada no soro dos pacientes após a ressecção cirúrgica do tumor permite sugerir papel potencial dessa proteína como biomarcador para a detecção precoce de recorrência do carcinoma epidermoide da cavidade bucal. A expressão tecidual da proteína MIF e seu receptor CD74 apresentou papel controverso, mas a concentração salivar da proteína MIF parece relacionar-se a um possível papel pró-tumoral em carcinoma epidermoide da cavidade bucal
INTRODUCTION. The Macrophage Migration Inhibitory Factor (MIF) overexpression is frequently observed in tumor tissues compared to normal tissues and some previous studies have suggested its role as a prognostic marker of malignancies, including hepatocellular, ovarian, esophageal and also head and neck carcinoma. Additionally, some of its mechanisms of action, as migration and cell proliferation induction, have been demonstrated, which allow imply a differential expression in tumor progression and therefore in the prognosis of malignant neoplasms. OBJECTIVES. This study aimed to evaluate the role of MIF protein and its receptor CD74 in prognosis and diagnostic of oral squamous cell carcinoma. METHODS. The study consisted of 50 patients with oral squamous cell carcinoma prospectively collected and 57 patients retrospectively collected admitted at the Head and Neck Surgery Service from Heliópolis Hospital and ABC Medical School. The analysis were performed by Imunohistochemistry of tumor and normal tissues and by ELISA of serum and saliva samples collected pre and post-surgical treatment. Results were correlated to clinical and histopathological data. RESULTS. The expression of MIF protein and of its receptor CD74 was higher in OSCC than in normal epithelium (p < 0,0001). Association between overexpression of MIF in tumor tissue and lymphatic vessel invasion was observed (p=0,005) and higher concentration of MIF in normal epithelium showed correlation of marginal significance with second primary tumor occurrence (p=0,072). The positive expression of the receptor CD74 did not presented association with clinical or histopathological variables. Serum MIF concentration presented inverse association with lymph node metastasis (p=0,018) and advanced pathological stage (p=0,040) and it was significantly reduced after the surgery (p=0,001). The salivary MIF concentration was not significantly reduced after the surgery, but it was associated with pT3 and pT4 stages (p=0,001) and advanced pathological findings (p=0,032). CONCLUSIONS. The results showing significant reduction of MIF concentration in post-surgical serum of patients suggest its potential role as a biomarker to early detection of oral squamous cell carcinoma recurrence. The MIF and CD74 expression presented controversial role, but the salivary concentration of MIF seems to develop a possible pro-tumoral role

Avaliou-se a influência da exposição ao flúor (F) através da água de beber, da velocidade de crescimento das unhas, da idade e do gênero na concentração deste elemento nas unhas das mãos e dos pés. Em adição, verificou-se a correlação entre as concentrações de F na saliva total, saliva do ducto da parótida e unhas das mãos e dos pés. Participaram do estudo 300 indivíduos, das faixas etárias de 3-7,14-20, 30-40 e 50-60 anos, residentes de cinco comunidades brasileiras, sendo duas no Estado de São Paulo (Pirajuí e Bauru, com água não fluoretada e 0,7 mgF/L na água de abastecimento, respectivamente) e três no Estado da Paraíba (Cajazeirinhas, Brejo dos Santos e Brejo das Freiras, com 0,2, 0,7 e 1,7 mgF/L na água de consumo, respectivamente). Foram coletadas duas ou três (apenas em Bauru) amostras de água de beber, além de duas amostras de: unhas dos pés, unhas das mãos, saliva total e saliva do ducto da parótida de cada indivíduo. O F nas amostras foi analisado com eletrodo íon-específico. Os dados obtidos foram analisados estatisticamente através de análise de variância e regressão linear (p<0,05). A exposição ao F através da água de beber, a velocidade de crescimento das unhas, a idade e o gênero influenciaram a concentração de F nas unhas das mãos e dos pés, sendo que o fator de exposição ao F através da água foi o que exerceu maior influência pelo modelo de regressão linear adotado. As unhas dos pés (R2=0,46) se mostraram melhores indicadoras de exposição ao F do que as das mãos (R2=0,24). Foi encontrada uma correlação negativa significativa entre velocidade de crescimento das unhas e concentração de F nas mesmas. Houve correlação positiva significativa entre concentração de F nas unhas das mãos e dos pés com: saliva total (r=0,36 e r=0,41) e saliva do ducto da parótida (r=0,25 e r=0,53), respectivamente. Também se observou correlação positiva entre saliva total e do ducto da parótida (r=0,24), bem como entre concentração de F na água e saliva total (r=0,41) e saliva do ducto (r=0,65). Todos os fatores testados influenciaram os níveis de F nas unhas e, portanto, devem ser levados em consideração quando se utiliza este marcador biológico.
The influence of fluoride (F) concentration in the drinking water, nails growth rate, age and gender upon the F content in fingernail and toenail were evaluated. In addition, the correlations among the F concentrations in whole saliva, parotid ductal saliva and finger/toenails were verified. Three hundred volunteers of 3-7, 14-20, 30-40, 50-60 years participated. They were residents of five Brazilian communities, two in Sao Paulo State (Pirajuí and Bauru, non-fluoridated and 0.7 mgF/L artificially fluoridated drinking water, respectively) and three in Paraiba State (Cajazeirinhas, Brejo dos Santos and Brejo das Freiras, 0.2, 0.7 and 1.73 mgF/L naturally fluoridated drinking water, respectively). Two or three samples of drinking water, and two samples of fingernails, toenails, whole saliva and ductal saliva were collected from each volunteer, with one-week interval period between the collections. F in water, whole saliva, ductal saliva and nails was determined using the ion-sensitive electrode. Data were analyzed by ANOVA and linear regression (p<0.05). The F exposure from the drinking water, nails growth rate, age and gender influenced the levels of F in fingernails and toenails. Considering the model of multivariate linear regression adopted, F exposure from the water influenced the most. Toenails (R2=0.46) seemed to be better indicators of F than fingernails (R2=0.24). It was found a significant negative correlation between nails growth rate and their content of F. Positive correlations were found between F concentration in fingernails and toenails and: F concentration in whole saliva (r=0.36 and r=0.41) and in parotid ductal saliva (r=0.25 and r=0.53), respectively. Moreover, it was observed a positive correlation between whole and parotid saliva (r=0.24), as well as between F concentration in the drinking water and whole (r=0.41) and parotid saliva (r=0.65). All factors that influenced nails F concentration must be taken into account when using them as biological markers.

The study of saliva for laboratory analyses is an increasing area of research with implications for basic and clinical purposes. Although this biological fluid is easy to manipulate and collect, attention must be directed to sample collection and storage, to method development and validation and to variability evaluation. The analysis of saliva provides important information about the functioning of various organs within the body. In this respect, endocrine research certainly occupies a central role. In effect, some hormones commonly measured in plasma, such as steroids, non-steroid, peptide and protein hormones, can be detected in the oral fluid. The protein polypeptide hormones are maybe a new analytical approach of the medicine laboratory but, at present time, there are still too few investigations about protein and polypeptide hormone levels in saliva. Detection of steroid hormones is perhaps the most interesting application in salivary hormonal studies. Steroids have often been studied because salivary-free steroid hormones seem to reflect the serum-free levels. Among of these steroids, salivary cortisol measurement is today a widely accepted alternative to the determination in plasma. In the section I of the present dissertation, a new ELISA method is explained. Research was carried out to study and validate an assay to measure salivary free IGF-I (sIGF-I) in human saliva. The detection range, the detection limit, the imprecision, the recovery and the specificity were evaluated. The pre-analytical variation was also studied. After the method validation, sIGF-I levels were measured in sedentary subjects and in athletes (protocol A and protocol C); moreover two different acute physical exercises in two groups of athletes were investigated to assess a possible effect on sIGF-I (protocol B and protocol C). The section II describes an analysis method which used the chromatographic technique. A SPE-HPLC method with UV detection was developed and validated to simultaneously measure cortisol (sF) and cortisone (sE) in human saliva. The analytical performances, in terms of detection range, sensitivity, imprecision, recovery, were evaluated. The pre-analytical variation, with respect to collection strategy and storage conditions, was also examined. After validation, the sF and sE method was applied analyzing specimens collected from athletes, before and after a physical exercise (protocol C). The results suggest further investigation from the laboratory point of view, taking into account the aspects related to the various forms and the specific and unspecific binding proteins (for sIGF-I assay) and to other steroid hormones and related metabolites identified and probably present in human saliva (for sF/sE assay).
Lo studio della saliva nelle analisi di laboratorio è un’area di ricerca in forte crescita, per le sue implicazioni nella ricerca di base ma anche a fini clinici. Sebbene questo fluido biologico sia facile da manipolare e da raccogliere, bisogna porre attenzione ai processi di raccolta e stoccaggio del campione, nonché allo sviluppo e alla validazione di metodi analitici, assieme alla valutazione delle variabilità. L’analisi della saliva dà importanti informazioni sul funzionamento di vari organi del corpo. In relazione a questo, la ricerca endocrina occupa certamente un ruolo centrale. Infatti, alcuni ormoni normalmente misurati nel plasma, come ormoni steroidei, ma anche ormoni non steroidei, peptidici e proteici, possono essere identificati nel fluido orale. Un nuovo approccio analitico nella medicina di laboratorio è forse rappresentato dagli ormoni polipeptidici e proteici ma, tuttora, ci sono ancora troppi pochi studi su questi ormoni salivari. La misura degli ormoni steroidi, invece, rappresenta forse l’applicazione più interessante negli studi degli ormoni salivari. Spesso gli steroidi sono studiati perché la concentrazione salivare riflette i livelli sierici. Tra i vari steroidi, la misura del cortisolo salivare è oggi una alternativa alla sua determinazione plasmatici. Nella I sezione viene spiegato un nuovo metodo ELISA. Sono stati sviluppati test sperimentali per studiare e validare un metodo per la misura dell’IGF-I libero salivare (sIGF-I). Sono stati studiati il range di misura, la sensibilità, l’imprecisione, il recupero e la specificità. Inoltre è stata studiata anche la variabilità pre-analitica. Dopo la validazione del metodo, sono stati misurati i livelli di sIGF-I in soggetti sedentari ed in atleti (protocollo A e protocollo C); inoltre è stato studiato il possibile effetto di due differenti esercizi fisici (in acuto) sulle concentrazioni di sIGF-I (protocollo B e protocollo C). La II sezione prende in esame un metodo di analisi che usa la tecnica cromatografia. E’ stato sviluppato e valicato un metodo SPE-HPLC con rivelazione UV per la misura contemporanea del cortisolo (sF) e del cortisone (sE) nella saliva umana. Sono state calcolate le performance analitiche (range di misura, sensibilità, imprecisione, recupero). E’ stata considerata anche la variabilità pre-analitica con particolare attenzione alle condizioni di raccolta e conservazione del campione. Dopo la validazione, questo metodo è stato applicato a campioni raccolti da un gruppo di atleti, prima e dopo un esercizio fisico (protocollo C). I risultati ottenuti suggeriscono ulteriori approfondimenti soprattutto da un punto di vista laboratoristico, tenendo presente la possibile presenza di varie forme e di specifiche ed aspecifiche proteine di legame (per sIGF-I) e altri ormoni steroidei e loro metaboliti identificati e probabilmente presenti nella saliva umana (per sF/sE).

No estudo das marcas de mordidas, os materiais biológicos relacionados à Odontologia, como o dente e saliva, são analisados por meio de evidências físicas (análise métrica e/ou emparelhamento físico) e evidências biológicas (análise de DNA). A partir de ambos os métodos pode-se estabelecer a identidade de um indivíduo, podendo assim, nas investigações forenses, apontar, ou mesmo identificar, um suspeito em uma cena de crime. O presente trabalho aplicou quatro métodos de análise física nas marcas de mordida produzidas em queijos e chocolates, bem como obter a recuperação de DNA, extraídos da saliva destes alimentos mordidos e de garrafas de água consumidas. Para tal, 20 participantes (10 do sexo masculino e 10 do sexo feminino) morderam cinco pedaços de queijo, cinco pedaços de chocolate e beberam em cinco garrafas de água, totalizando 15 amostras para cada um. As amostras produzidas foram armazenadas e analisadas em diferentes intervalos de temperatura (ambiente e geladeira) e tempo (imediato, três dias e sete dias). Na análise física, os métodos aplicados foram: análise métrica utilizando um paquímetro digital (Digital Caliper, Cixi Xinzheng Trade Co., Ltd., Zhejiang, China); análise métrica utilizando o software ImageJ (National Institutes of Health, Bethesda, Maryland); sobreposição manual; e a sobreposição digital por meio do software Adobe Photoshop (Adobe Systems, Inc., Mountain View,California, USA). Na análise biológica foram realizadas as seguintes etapas do exame de DNA: coleta da saliva pela técnica de duplo swab, extração do DNA utilizando Kit QIAamp (Qiagen, Hilden, Germany), quantificação do DNA recuperado através do equipamento espectofotômetro Nanodrop (Thermo Scientific™, Wilmington, DE, USA), amplificação dos marcadores utilizando o Kit de identificação humana AmpFLSTR Identifiler PCR Amplification (Applied Biosystems®, Carlsbad, CA, EUA) e a eletroforese em gel de agarose. De acordo com os resultados encontrados, não houve diferença significativa entre os dois métodos de análise métrica. Na análise de emparelhamento físico, o método manual obteve o maior número de identificados, com 58% para ambos os sexos, enquanto o método Adobe Photoshop apenas 32% das amostras foram identificadas para o sexo feminino e 44% no sexo masculino. O valor do índice de concordância intraobservador foi classificado como substancial para os métodos manuais em ambos os sexos e para o método Adobe Photoshop para o sexo masculino; e como moderado para o método Adobe Photoshop para o sexo feminino. Nas evidências biológicas, as amostras de DNA coletadas a partir da saliva depositada em queijos e chocolates mordidos e garrafas de água bebidas tiveram valores de concentração variando entre 28,52 ± 14,00 a 8,99 ± 2,20 ng/μl, e estas foram suficientes para amplificação. Com isso, conclui-se que amostras de águas, queijos e chocolates nas condições estudadas, que simularam alimentos mordidos e bebidas consumidas encontrados em cenas de crime ou armazenadas em geladeiras a espera da análise pericial, podem ser utilizadas nas investigações de identificação em marcas de mordida tanto para métodos de evidências físicas quanto para métodos de evidências biológicas.
In the study of bite marks, the biological materials related to dentistry, such as teeth and saliva, are analyzed by means of physical evidence (metric analysis and / or physical pairing) and biological evidence (DNA analysis). From both methods can establish the identity of an individual, thus allowing, in forensic investigations, point, or even identify a suspect in a crime scene. This study aimed to apply four methods of physical analysis in bite marks produced in cheeses and chocolates, as well as obtain recovery the DNA extracted from the saliva of bitten food and bottled water consumed. The sample was comprised of 20 volunteers, 10 males and 10 females. Each volunteer was instructed to bite five pieces of cheese, five pieces of chocolate and drink five bottles of water, producing 15 samples of each from the 20 participants. The produced samples were stored and analyzed in different temperature ranges - temperature (25°C) and refrigerator (4-8°C), and time (immediately, three days, seven days). The methods used were: metric analysis by using a digital caliper, digital metric analysis using ImageJ software (National Institutes of Health, Bethesda, Maryland); manual overlay of the plaster model of the dental arches versus plaster model of chewed food, and overlapping images using Adobe Photoshop software (Adobe Systems, Inc., Mountain View,California, USA). In biological testing, saliva collecting was done using the double swab technique, DNA extraction according to the extraction protocol of the QIAamp kit (Qiagen®, Hilden, Germany), quantification of recovered DNA using equipment Nanodrop spectrophotometer (Thermo Scientific™, Wilmington, DE, USA); amplification of the markers was performed by human identification kit Identifiler PCR Amplification AmpFLSTR (Applied Biosystems®, Carlsbad, CA, EUA) and agarose gel electrophoresis. According to the results, there was no significant difference between the two methods of metric analysis. In the physical pairing analysis, the manual method had the highest number of identified subjects with 58% for both sexes while with the Adobe Photoshop method only 32% of the samples were identified for females and 44 % males. The index value for intraobserver agreement was rated substantial for manual methods in both sexes and for Adobe Photoshop method for males; and as moderate for Adobe Photoshop method for females.In biological evidence, samples of DNA from saliva deposited in cheese and bitten chocolate, beverage and water bottles had concentration values ranging from 28,52 ± 14,00 a 8,99 ± 2,20 ng/μl, and these were sufficient for amplification. Thus, it was concluded that samples of water, cheeses and chocolates under the conditions studied, simulating bitten foods and beverages found at crime scenes or stored in refrigerators waiting for the forensic analysis can be used for investigations in identifying bite marks using both physical methods evidence as to biological method evidence.

Koffein (1,3,7-Trimethylxanthin) und Coprostanol (5beta-cholestan-3beta-ol) wurden im Berliner Oberflächenwasser nachgewiesen. Ihre Konzentrationen korrelierten mit dem Verunreinigungsgrad der Proben, was nahelegt, dass sie sich als Marker für menschliche Aktivität eignen. Bemerkenswerterweise wurde Koffein in jeder einzelnen Oberflächenwasserprobe oberhalb der Bestimmungsgrenze von 0,025 µg/L gefunden. Um Oberflächenwasserproben in größeren Serien zu untersuchen, war die Entwicklung zweier neuer Methoden erforderlich: ein Immunoassay, basierend auf einem monoklonalen Antikörper für Koffein und eine dispersive flüssig-flüssig Mikroextraktionsmethode (DLLME), gefolgt von Flüssigkeitschromatographie gekoppelt mit Tandem-Massenspektrometrie (LC-MS/MS) für Coprostanol. Der entwickelte Koffein-Immunoassay zeigt die beste je erhaltene Nachweisgrenze für Koffein (0,001 µg/L), erlaubt Hochdurchsatz-Analysen und erfordert keine Probenvorbereitung. Der Assay wurde auch erfolgreich für die Messung von Koffein in Getränken, Haarwaschmitteln, Koffeintabletten und menschlichem Speichel angewendet. Antikörper gegen Coprostanol sind nicht kommerziell erhältlich. Eine neue Strategie Anti-Coprostanol-Antikörper zu generieren wurde erarbeitet, die eine analoge Verbindung – Isolithocholsäure (ILA) – als Hapten verwendet, mit der eine Gruppe von Mäusen immunisiert wurde. Ein polyklonales Anti-ILA-Serum wurde produziert, welches Coprostanol bindet, aber die niedrige Affinität erlaubte nicht den Aufbau eines Immunoassays, der die Messung von Umweltkonzentrationen des Anayten (im Bereich ng/L) zulässt. Spezifische Anti-ILA-Immunglobuline G wurden auch in den Faeces der Mäuse gefunden. Coprostanol wurde in den Wasserproben durch die Verwendung einer neuentwickelten LC-MS/MS-Methode unter APCI-Ionisation (atmospheric pressure chemical ionisation) gemessen. Konzentrationen oberhalb von 0,1 µg/L wurden nach Voranreicherung der Probe mittels DLLME bestimmt.
Caffeine (1,3,7-trimethylxanthine) and coprostanol (5beta-cholestan-3beta-ol) were detected in samples of Berlin’s surface water. Their concentrations correlated with the contamination status of the samples, suggesting their usefulness as markers of human activity. Remarkably, caffeine concentrations were always well above the limit of quantitation of 0.025 µg/L. In order to screen surface water samples in larger series, the development of two novel methods was required: a monoclonal antibody-based immunoassay for caffeine and a dispersive liquid-liquid microextraction (DLLME) method, followed by liquid chromatography tandem mass spectrometry (LC-MS/MS) for coprostanol. The caffeine immunoassay developed shows the best analytical limit of detection (LOD) obtained so far for caffeine (0.001 µg/L), allows high-throughput analysis, and does not require sample pre-treatment. The assay was also successfully employed to measure caffeine in beverages, shampoos, caffeine tab-lets, and human saliva. Antibodies to coprostanol are not commercially available. A new strategy to generate anti-coprostanol antibodies was elaborated using an analogous com-pound as hapten – isolithocholic acid (ILA) – and immunizing a group of mice. A polyclonal anti-ILA serum was produced, which binds coprostanol but the low affinity did not permit setting up an immunoassay to measure environmental concentrations of the analyte (in the range of ng/L). Specific anti-ILA immunoglobulin G were also found in the faeces of the immunized mice. Coprostanol was quantified in the water samples using a newly developed LC-MS/MS method using atmospheric pressure chemical ionisation (APCI). Concentrations above 0.1 µg/L were determined after sample preconcentration using DLLME. This extraction method also proved to be successful for enrichment of coprostanol-related compounds such as cholesterol, cholestanol, cholestanone, ergosterol, and stigmasterol.

Migraine is a disabling neurological disorder with high prevalence, the clinical manifestations of which are highly dependent on stress. The overall theme of the present thesis was to address aspects of stress in migraine. A multimodal behavioral treatment (MBT) program was developed specifically designed for migraine and focusing on stress as a trigger and an intervention was performed using this Internet-administered program. Migraine symptoms were followed via an Internet administered diary and questionnaires were answered at regular intervals during the 11-month study period. The thesis is based on four papers: In Paper I, life events and current stress, personality traits, and gender were studied cross-sectionally in 106 women and 44 men with migraine, who suffered at least two attacks a month at inclusion. Paper II describes a randomized controlled trial of the MBT program performed on 58 women and 25 men recruited from participants of the study described in Paper I. In the MBT study participants were randomized into one control group and two MBT groups, one of which received hand massage as part of the treatment. In Paper III, complete migraine drug use and changes in use and in drug efficacy during the MBT program were studied. In Paper IV, the salivary cortisol levels of MBT participants were evaluated as a biological stress marker. The MBT program proved effective in decreasing migraine headache; it was feasible and there was low attrition. Moreover, MBT resulted in decreased migraine drug use and increased drug efficacy, but had no discernible effects on salivary cortisol profiles. No effect of hand massage on migraine headache frequency was seen. Personality trait profiling revealed high scores for the neuroticism factor. Stress susceptibility was the single most aberrant personality trait and correlated highly with the reported level of current stress and with experienced negative life events. Gender differences included higher scores for women on trait anxiety, negative life events, depressive mood, anxiety, tension type headache, use of triptans, and efficacy of analgesics, whereas men displayed higher use of analgesics. In conclusion, the efficacy and low attrition associated with the present MBT program appears promising and timely with regard to the development of better and more accessible migraine treatment. Stress susceptibility, gender, negative life events and psychosomatic comorbidity are important factors to consider in relation to the care of persons with migraine.

Bakgrund: Gingivit kännetecknas av svullet/lättblödande och ibland ömt tandkött. Gingivit är en inflammatoriskt orsakad reaktion som uppkommer i samband med närvaro av bakterier. För att motverka utvecklingen av gingivit rekommenderas munhygienvård regelbundet. Gingivit kan utvecklas till parodontit med skador på upphängningsapparaten och benstödet, orsaken till detta är idag dock ej helt klarlagd. Kliniska variabler (blödning vid sondering) är vedertagna diagnostiska metoderna för diagnostisk av gingivit idag. I pilotstudier har laktoferrinkoncentrationer setts vara förhöjda i stimulerad helsaliv och gingivalexsudat hos patienter med kronisk parodontit jämfört med patienter utan parodontit. Mål: Undersöka hur koncentrationen laktoferrin i ostimulerad helsaliv förändrades hos individer som utvecklade gingivit i en experimentell gingivitstudie och om C-reaktivt protein är en tillförlitlig markör för gingival inflammationsgrad.Material och metod: En experimentell gingivitstudie genomfördes med 13 försöksdeltagare där munhygienvanor kontrollerades och förändrades under 3 perioder á 2 veckor. Helsalivprov och mätning av CRP genomfördes. Resultat: Gingivalindex och CRP höjdes signifikant under försöksperioden Dag 0 till Dag 13(p=0,002 och p=0,027). Laktoferrin sågs först signifikant förhöjd under mitten av avslutningsperioden (Dag 20) (p=0,015). Laktoferrinkoncentrationen återgick inte till baselinevärdet under studien. Signifikant korrelation sågs mellan gingivalindex och laktoferrin från Dag 0 fram till Dag 13 (r=0,224, p=0,042). Utvecklingen av CRP sågs inte korrelera med någon variabel under försöket. Konklusion: Resultaten indikerar att utvecklingen av experimentell gingivit påverkar laktoferrinkoncentrationen över tid och kan därför vara en potentiell biomarkör för gingivit. De låga skillnader hos CRP visar att den inte är en tillförlitlig markör vid gingivit.
Background: Gingivitis is characterized by swollen and bleeding tissue, an inflammatory reaction with presence of pathological bacteria. To counteract the development of gingivitis, regular oral hygiene care is recommended. Gingivitis can progress into periodontitis which damages the periodontal tissue and bone. Clinical variables (bleeding on probing, plaque occurrence) are diagnostic methods for gingivitis. Elevated concentrations of lactoferrin in stimulated whole saliva and gingival crevicular fluid has been seen in patients with chronic periodontitis compared to patients without periodontitis.Objectives: Investigate the salivary concentration of lactoferrin in subjects enrolled in the experimental gingivitis model, the response of capillary CRP was also investigated.Methods: Thirteen individuals participated in a 14-day experimental gingivitis model and 10-day gingival healing period, measurements of whole saliva lactoferrin and CRP was performed. Results: Gingival index and CRP was increased statistical significant during the induction of gingivitis (p=0.002 and p=0,027). Lactoferrin increased during the experimental phase and reached a peak at day 20, which was statistical significant compared to baseline (p=0.015). A statistical significant correlation between gingival index and lactoferrin was seen from day 0 to day 13 (r=0.224, p=0.042). CRP did not match any correlation to any other measured variable from day 0 to day 24.Conclusion: These results indicate that gingivitis may have a systemic effect, which in this study has been expressed as increased levels of salivary lactoferrin. Lactoferrin may be a potential biomarker for gingivitis. Due to low changes in concentrations and because CRP is systemically affected it makes an unreliable biomarker for gingivitis.

La dengue est une maladie virale des régions tropicales et subtropicales, transmise par les moustiques du genre Aedes. Le virus de la dengue (DENV) appartient à la famille des Flaviviridae, genre Flavivirus. Si la plupart des infections sont asymptomatiques ou se traduisent par un syndrome fébrile sans gravité, le virus peut aussi causer une maladie plus sévère caractérisée par une fuite plasmatique, avec ou sans hémorragie. Sans prise en charge adéquate, les formes les plus sévères peuvent évoluer vers un syndrome de choc, potentiellement mortel. Il n’existe pas de traitement spécifique de la dengue mais une réhydratation adaptée et débutée précocement permet de réduire la survenue de formes sévères de la maladie. Malheureusement, les symptômes initiaux de la dengue avant la survenue des éventuelles complications ne sont pas spécifiques et seul un diagnostic biologique basé sur la détection du génome viral, de l’antigène NS1 ou des anticorps anti-DENV dans le sang des patients permet de confirmer la nature exacte de l’infection. La dengue constitue à l’heure actuelle un problème majeur de santé publique du fait de son expansion mondiale et de l’augmentation annuelle du nombre de cas sévères. Pour assurer la surveillance épidémiologique et le contrôle de la maladie, il est indispensable de développer des outils diagnostiques performants et faciles à mettre en œuvre, à la fois utilisables par les médecins de toutes les structures médicales, des simples centres de soins de santé primaire aux centres de référence, et utilisables lors d’enquêtes épidémiologiques pour l’investigation de nouvelles épidémies. Le travail de cette thèse a porté sur plusieurs aspects de cette problématique. Dans une première partie, un test commercial de diagnostic rapide (TDR) permettant la détection simultanée de la NS1 et des IgG et IgM anti-DENV, a été évalué, en laboratoire spécialisé et sur le terrain, afin de comparer, à partir des mêmes échantillons, les performances du test dans deux situations différentes. La sensibilité s’est révélée plus faible lors de l’utilisation sur le terrain que lors de l’utilisation en laboratoire de référence. La majorité des discordances a été observées pour la détection des IgG et des IgM. L’impact de la mise à disposition du test sur la prise en charge des patients a également été évalué et il s’est avéré qu’au cours de cette étude les pédiatres cambodgiens ont ignorés les résultats du test rapide et ont préféré suivre leur instinct clinique.Un second volet a porté sur la faisabilité d’utiliser les urines et la salive en remplacement du sang veineux pour les tests employés en routine pour le diagnostic de la dengue. Les urines et la salive sont des fluides biologiques plus faciles à prélever que le sang veineux ce qui présente un avantage majeur pour les enquêtes épidémiologiques mais peut également secourir les médecins lorsqu’un prélèvement de sang veineux est difficile à obtenir, par exemple chez les enfants. Bien que les performances des différentes méthodes de diagnostic ne soient pas aussi bonnes avec de l’urine et la salive qu’avec du plasma, les résultats obtenus par PCR en temps réel et avec les ELISAs de détection des anticorps anti-DENV démontrent l’intérêt potentiel de ces deux fluides biologiques pour détecter les infections par le DENV lorsqu’il est difficile d’obtenir du sang veineux. Plusieurs TDR commerciaux développés pour permettre la détection de la NS1 et des anticorps anti-DENV (IgM, IgG et IgA) dans les urines et la salive ont été évalués mais les performances obtenues se sont révélées peu satisfaisantes.Une dernière partie du travail a été consacrée à l’étude de la protéinurie comme marqueur pronostic potentiel de sévérité de la dengue. Ce marqueur biologique ne s’est pas révélé être utile pour diagnostiquer précocement les formes sévères de la maladie
Dengue is a viral disease transmitted by Aedes species mosquitoes, in tropical and subtropical regions. Dengue virus (DENV) belongs to the family Flaviviridae, genus Flavivirus. Although most DENV infections are asymptomatic or result in a self-limited febrile illness, severe diseases characterized by plasma leakage, with or without hemorrhage, can also occur. Patients with a severe dengue can rapidly progress into a life-threatening shock syndrome if no efficient clinical management is provided. There is no specific treatment available for dengue but an accurate and early fluid therapy substantially reduces the occurrence of severe forms of the disease. Dengue symptoms are typically non-specific until or unless complications develop. Only a biologic diagnosis based on DENV genome, NS1 antigen or anti-DENV antibodies detection enables to confirm dengue cases. Dengue is now a major public health problem due to both its geographical spread and the increase in the number of severe cases. New diagnostic tools are necessary to ensure epidemiological surveillance and control of the disease. These tools need to be effective and easy to use in every medical settings, from the smallest primary health centers to the biggest reference centers, and also usable for epidemiologic studies, e.g. for epidemic investigations. The work presented in this thesis was dedicated to this problematic.In a first part of the work, a rapid diagnostic test (RDT), designed to detect NS1 antigen, anti-DENV IgG and IgM, was evaluated, both in a specialized laboratory and in the field, in order to compare the test performances in two different settings, with the same samples. Interestingly, sensitivity was lower when the test was used in the field compared to the sensitivity of the test when performed in the specialized laboratory. Discordances were mainly observed for IgM and IgG detection. Impact of the use of the RDT on clinical management was also assessed during the field study and it revealed that Cambodian pediatricians ignored the results of the RDT and followed their clinical instinct.A second part of the work was dedicated to the assessment of the usefulness of urine and saliva for dengue diagnostic. Dengue diagnostic normally requires a venous blood sample that can be difficult to obtain in certain conditions such as in children or during epidemiological studies. Urine and saliva are easier to collect as the procedure is non-invasive. We showed that, although the performances of the different diagnostic methods were not as good in saliva and urine as in plasma specimens, the results obtained by qRT-PCR and by anti-DENV antibody ELISA could well justify the use of these two body fluids to detect dengue infection in situations when the collection of blood samples is difficult. Performances of commercial RDTs developed for NS1 and anti-DENV antibodies (IgM, IgG and IgA) detection in urine and saliva specimens were not satisfactory.In the last part of the thesis, the potential use of proteinuria as a prognostic marker of severity was assessed but it didn’t prove to be a useful marker for risk prediction

Eviter la piqûre de moustiques vecteurs en utilisant des mesures antivectorielles reste le meilleur moyen de se protéger des maladies vectorielles. La salive de moustique peut induire une réponse anticorps (Acs) spécifique chez l’hôte qui pourrait être utilisé pour définir l'efficacité de ces mesures de protection antivectorielle. L’objectif de notre projet était d’évaluer la possibilité d’utiliser cette réponse Acs anti-salive de moustiques pour mesurer l’exposition à des espèces spécifiques de moustiques ainsi que d’identifier des marqueurs d’exposition. Nous nous sommes tout d’abord assurés de l’absence de différences intraspécifiques entre différentes colonies de moustiques, une condition indispensable pour pouvoir observer des différences au niveau de l’espèce. Par ailleurs, nous avons mis au point un protocole pour préserver les échantillons salivaires dans des conditions de terrains non optimales. A partir de ces expérimentations préliminaires, nous avons évalué la diversité du répertoire protéique salivaire de quatre espèces d’Anopheles par des différentes approches, et montré une spécificité de genre et d’espèce aussi bien au niveau protéique qu’antigénique. Enfin, nous avons montré une évolution spatio-temporelle de l’intensité de la réponse Acs anti-salive ainsi que sa spécificité de genre et d’espèce, chez des individus exposés à différents niveaux à Ae. caspius. Ces résultats souligne la possibilité de caractériser des antigènes salivaires spécifiques de genre et d’espèces qui peuvent avoir un intérêt pour mesurer le contact hôte/vecteur au niveau individuel, le risque de transmission de maladies vectorielles ou l’efficacité des mesures antivectorielles
The primary mean to protect individuals from arthropod-borne diseases is the prevention of bites from infected arthropods which could be achieved by vector control strategies. Mosquito saliva could induce a specific antibody response in exposed individuals that could be used to assess the effectiveness of anti-vector measures. The aim of this study is to assess the possibility to use anti-mosquito saliva antibody responses in order to evaluate the exposure to specific species of vectors and to identify salivary protein candidates that can be used as immunological markers of exposure. We first verify the lack of intraspecific differences among several mosquito colonies which is essential to further observe potential differences at the species level. Moreover, a convenient storage method was developed to preserve salivary samples in non optimal condition on the field. Based on these preliminary results, we evaluated the salivary gland protein repertory diversity among four Anopheles species using complementary approaches and we shown a genus and species specificity at the protein and antigen level. At least, a spatio-temporal evolution of anti-saliva antibody responses was shown according to the Aedes caspius density using sera of differentially exposed individuals. The specificity of this response was also reported at the genus and species level. All together, these results suggest the feasibility to characterize genus and species specific salivary antigens which could be used as immunological markers of exposure to evaluate host/vector contacts, the risk of vector-borne disease transmission or the effectiveness of anti-vector strategies

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The regional lymph nodes play a pivotal role in diagnosis, staging and management of head and neck squamous cell carcinomas (HNSCC). Despite their importance, detailed understanding of the probable mechanisms of lymphatic metastases has not been completely achieved. Subjects and Methods: We analyzed metastatic and normal lymph node tissues, as well as saliva and serum from sixth-two patients with HNSCC, and twenty-nine controls using two-dimensional electrophoresis, MALDI-Q-TOF and western blot. Results: Several proteins were found to be significantly increased in metastatic nodes, such as stratifin, glutathione S-transferase pi, apoliproteín A-I, alpha-1-microglobulin, disulfide isomerase, galectin, citokeratins, immunoglobulins, transtirretin, calciun-binding protein (família S100) and fat-binding protein (FABP). Among the down-regulated proteins in metastatic lymph nodes are calreticulin, tropomiosin 3, triosephosphate isomerase, piruvate quinase, anidrase carbonic, gamma actin, peroxiredoxin 2, profilin 1, gliceraldeyde 3- fosfato desidrogenase and heat shock proteins. These proteins are involved in epidermis development, cell proliferation, migration and adhesion, apoptosis, defense and inflammatory response and xenobiotic metabolism. Our data on the expression of heat shock proteins and enzymes of the glycolytic pathway suggest an effect of the lymph node environment in controlling tumor progression or in metabolic reprogramming of the metastatic cell. In saliva, 13 proteins showed an altered pattern of expression in samples patient, including over-expression of keratins, immunoglobulins, alphaamylase, PLUNC and zinc-alpha-2-glycoprotein and down-regulation of myosin. In serum samples, six proteins were over-expressed (serum albumin, alpha-1- microglobulin/bikunin precursor, apolipoprotein A-I, haptoglobin, serotransferrin, transthyretin) and two were under-expressed (hemoglobin subunit alpha, hemoglobin subunit beta) compared to the control group. Conclusion: New potential markers, such as profilin-1 and E-FABP, were identified and may be proved useful for defining the invasive phenotype of head and neck carcinomas.
O comprometimento de linfonodos regionais por células neoplásicas é atualmente o indicador mais utilizado para prognóstico em pacientes com carcinoma epidermóide de cabeça e pescoço (CECP). Apesar disso, a compreensão detalhada dos mecanismos envolvidos na formação de metástases linfáticas ainda não foi completamente atingida. Casuística e Método: Foi avaliado o perfil protéico de linfonodos metastáticos e não metastáticos, bem como de amostras de saliva e soro de 62 pacientes em diferentes estágios da doença e de 29 controles, utilizando eletroforese bidimensional, espectrometria de massas por MALDI-Q-TOF e experimentos de validação por Western blot. Resultados: Os resultados mostraram várias proteínas com expressão elevada em linfonodos metastáticos em relação aos não metastáticos, como stratifina, glutathiona S-transferase pi, apoliproteína A-I, alfa-1-microglobulina, dissulfeto isomerase, galectinas, citoqueratinas, imunoglobulinas, transtirretina e proteínas de ligação ao cálcio (família S100) e a ácidos graxos (FABP). De forma inversa, as proteínas calrreticulina, tropomiosina 3, triofosfato isomerase, piruvato quinase, anidrase carbônica, gama actina, peroxirredoxina 2, profilina 1, gliceraldeído 3-fosfato desidrogenase e proteínas de choque térmico mostraram níveis reduzidos em linfonodos metastáticos. Essas proteínas estão envolvidas em processos de desenvolvimento epidérmico, proliferação, migração e adesão celular, apoptose, resposta inflamatória e metabolismo de xenobióticos. Os dados relacionados à expressão de proteínas de choque térmico e enzimas da via glicolítica sugerem um efeito do ambiente dos linfonodos e no controle da progressão do tumor ou na reprogramação das células metastáticas. Em saliva, 13 proteínas exibiram um padrão alterado nas amostras de pacientes com câncer, incluindo expressão elevada de queratinas, imunoglobulinas, alfa-amilase, PLUNC e zinc-alfa-2-glicoproteína e expressão reduzida de miosina. Em amostras de soro, seis proteínas apresentaram expressão aumentada (albumina, alfa-1-microglobulina/bikunina precursor, apolipoproteína A-I, haptoglobina, serotransferrina e transtirretina) e duas estavam com expressão diminuída (hemoglobina alfa e hemoglobina beta), quando comparadas com o grupo controle. Conclusão: Os resultados obtidos revelaram novos marcadores potenciais, como profilina 1 e E-FABP, PLUNC e transtirretin que podem ser úteis na definição do fenótipo invasivo e no rastreamento e diagnóstico desse grupo de neoplasias.

A research report submitted to the Faculty of Health Sciences, University of the Witwatersrand, in fulfilment of the research requirements for the degree of Master of Dentistry (MDent) in the field of Oral Pathology Johannesburg, 2015
Cell proliferation is associated with tumour biological behaviour. Correlation of proliferation marker and p53 expression with histologic grade in salivary gland neoplasia is important to establish tumour behaviour and prognostic biomarkers may be useful in their histologic differentiation and treatment. Objectives: To evaluate expression of proliferation markers MCM2 and Ki-67, and tumour suppressor gene p53 in salivary gland neoplasms and to correlate this expression with tumour type. Materials and methods: Tissue from 19 pleomorphic adenomas (PAs), 15 polymorphous low grade adenocarcinomas (PLGAs), 11 mucoepidermoid carcinomas (MECs), 12 acinic cell carcinomas (AcCCs) and 13 adenoid cystic carcinomas (AdCCs) was analysed for immunohistochemical expression of MCM2, Ki-67 and p53. The labelling index (LI) for each tumour was determined by counting the percentage of positive cells per 1000 tumour cells. A Kruskal-Wallis test was used to assess differences in LIs. Results: Overall, MCM2 (p=0.0001) and Ki-67 (p=0.0001) expression was significantly higher compared to p53 (p=0.2447) amongst the five salivary gland tumours. The AdCC MCM2 LI was significantly higher compared to AcCC (p=0.0024), PLGA (p=0.0002), MEC (p=0.0028) and PA (p=0.0001). There were no significant differences in MCM-2 expression between the other neoplasms. Conclusion: MCM2 is a more sensitive marker than Ki-67 and showed significantly greater expression in all tumours studied. The Ki-67 and MCM2 labelling indices were significantly higher in AdCC than in MEC, AcCC, PA and PLGA.

Dissertação de Mestrado em Química apresentada à Faculdade de Ciências e Tecnologia
A quimiometria é uma área da química cujas técnicas e metodologias têm uma aplicação muito ampla. O tratamento de dados através dos métodos quimiométricos permite uma análise que combina profundidade com clareza, após uma seleção judiciosa dos métodos a aplicar. Técnicas como a análise de agrupamentos hierárquica (HCA) e análise de componentes principais (PCA) são extremamente úteis no tratamento de dados multivariados, permitindo revelar a sua estrutura, muitas vezes recorrendo a representações gráficas adequadas, e sendo adequados mesmo quando a informação é muito vasta. Nesta dissertação, utilizaremos técnicas quimiométricas para a análise de conjuntos de dados bioquímicos e cineantropometricos relativos a atletas de escalões jovens nas modalidades de natação e futebol.Os resultados mostram que a conjugação dos métodos PCA e HCA permitem, entre vários outros aspetos, mapear o desempenho em função de características cineantropométricas dos atletas da modalidade de natação, e promover uma representação gráfica muito útil em que o conjunto de todos os atletas pode surgir a duas dimensões. Fez-se também a análise de parâmetros bioquímicos em relacionamento com cargas de treino aplicadas a atletas desta mesma modalidade, tendo sido patente a dificuldade de apresentar padrões bem definidos ou relações claras. Em contraste, atletas de escalões jovens do futebol, mostram perfis de indicadores bioquímicos mais regulares que permitem estabelecer, com facilidade, as caraterísticas e comportamentos em cada escalão.
Chemometrics is an area of chemistry whose techniques and methodologies have a very wide application. The treatment of data through chemometric methods allows an analysis that combines depth with clarity, through a judicious selection of methods to be applied. Techniques such as analysis hierarchical clustering (HCA) and analysis principal component (PCA) are extremely useful in the treatment of multivariate data, allowing to reveal their structure, often using appropriate graphical representations, and being adequate even when the information is very wide. In this dissertation, we will use chemometrics techniques for the analysis of biochemical and kinanthropometry data sets related to young athletes in swimming and football.The results show that the combination of analysis principal component and analysis hierarchical clustering methods allows, among several other aspects, to map the performance in function of kinanthropometry characteristics from the athletes that practice swimming as modality, and to promote a very useful graphical representation in which the set of all athletes can appear in two dimensions. The analysis of biochemical parameters was also carried out in relation to training the overload applied to athletes of this same modality, and the difficulty of presenting well-defined patterns or clear relationships was evident. In contrast, young footballers show more regular biochemical indicator profiles that allowed to easily establish the characteristics and behaviours in each class.

博士
國立臺灣大學
醫學工程學研究所
101
The main purpose in this dissertation is to measure the presence of the tumor marker carbohydrate antigen 15-3 (CA15-3) in human saliva using two different surface plasmon resonance (SPR) systems. To compare the sensitivity of an SPR biosensor based on thin-film Au/ZnO and the Biacore SPR system. CA15-3 is a tumor marker in human cancers, especially for the diagnosis of breast cancer recurrence and monitoring. It is used as a reference marker, or “diagnostic gold standard,” to which other breast cancer markers are compared. Low level of CA15-3 can be detected in saliva, and correlated with serum concentration closely. There are several advantages to detect CA15-3 in saliva such as: 1.Easy collection. 2. Repeatable without pain. 3. Reflect real time status of body status. There are also several difficulties still need to be overcome such as: 1.The variance of saliva detection in different collection methods. 2. The level of CA15-3 in saliva is up to 10-fold lower than that in serum. 3. To our knowledge, there are no commercially available salivary CA15-3 standards or controls to evaluate the performance of this detection method. In this study, we have to prepare varying concentrations of salivary CA15-3 samples at physiologically relevant concentrations. 4. It is unclear about processing variance for saliva CA15-3 detection. In this study, we prepared CA15-3 samples in saliva by matrix replacement method and analyzed intensity responses to the samples at various concentrations of CA15-3.in saliva by the Biacore SPR system and Au/ZnO thin film SPR system. The linear detection range with the SPR system based on thin-film Au/ZnO was 2.5–20 U/mL (the cut-off point in cancer patients is around 4 U/mL). The linear range with the Biacore SPR system was 40–300 U/mL. Previous reports from the Streckfus Lab have shown that the saliva CA15-3 concentration for healthy controls is about 3.19 ± 0.52 U/mL; for benign breast tumors, about 7.23 ± 1.58 U/mL; and for malignant breast cancer tumors, about 10.90 ± 3.44 U/mL. The Au/ZnO thin film SPR sensor system used in this study showed a linear response to CA15-3 across this entire concentration range. The KD values for the Biacore and our Au/ZnO chip were calculated to be 381 and 17 U/mL, respectively. Obviously, the KD of the Biacore system was much higher than that of our Au/ZnO SPR chip. This difference is mainly due to the limitations of the dextran matrix to present the probe in a homogeneous conformation with its target. CM5 matrix has intrinsic limitations in terms of steric hindrance, heterogeneities in the density and conformation of probes inside the gel, and kinetic resistance due to improper molecular orientation and mass transfer that cause an apparent decrease in the reaction-rate constant. Thus, this newly developed thin film SPR system is expected to be quite useful in the clinical diagnosis of cancer progression. These results show that thin-film Au/ZnO-based SPR systems have higher sensitivity and can be used for measuring the levels of CA15-3 in human saliva without concentrating the samples .

碩士
國立臺灣大學
職業醫學與工業衛生研究所
105
As the blossom of semiconductor industry, arsenic was used widely in the fab manufacturing process due to its metalloid properties, which therefore led to the concern of exposure hazard to workers on site. In order to characterize the association between inorganic arsenic exposure and work task, this study was conducted using saliva arsenic as biomarker to explore its association with the affecting factors. This study was incorporated into a LED manufacturing company’s annual physical examination program, and recruited a total of 355 subjects from this company’s northern and southern factories. Questionnaire was used to collect information on demographics, living habits, and seafood consumption. There were two parts in statistical analysis for the association between saliva arsenic concentration and job title. In the first part, study subjects were grouped as exposed group, control I, and II groups, based on the judgement of the occupational nurse of this company. In the second part, we combined job title along with arsenic exposure information collected using questionnaire to divide the study subjects into exposed group, low exposed group, and administration group. This study used inductively coupled plasma mass spectrometry (ICP-MS) to analyze arsenic concentration in the saliva samples of the study subjects. Results showed that average saliva arsenic concentration of all subjects was 0.66 μg/L, average saliva arsenic concentration of subjects with and without occupational arsenic exposure was 0.65 μg/L, and 0.60 μg/L, respectively. Though there was no significant association between saliva arsenic level and job title, there were positive correlations of saliva arsenic concentration with work seniority (r= 0.158, p< 0.01), workers’ age (r= 0.126, p< 0.05), alcohol drinking (r= 0.142, p< V 0.01), and betel nut chewing (r= 0.146, p< 0.01), respectively. However, saliva arsenic concentration significantly decreased with higher education level. In this study, significantly positive association of saliva arsenic level with work seniority and workers’ age suggested age-related factors were important to saliva arsenic concentration. Besides, negative association of saliva arsenic concentration with higher education level could be attributed to that study subjects with higher education level would work at higher job title, with relatively low arsenic exposure. Positive relationship between saliva arsenic concentration and living habits such as smoking, alcohol drinking, betel nut chewing was in accordance with previous studies’ finding. No significant association between saliva arsenic concentration and job title could probably be attributed to that various work tasks under the same job title might cause the misclassification of arsenic exposure based on job title. Further study with precise and detailed arsenic exposure history is anticipated in order to classify the relationship of saliva arsenic concentration, as biological marker, with worker’s occupational arsenic exposure.

碩士
國立臺灣大學
職業醫學與工業衛生研究所
104
Biomonitoring is the measurement of chemicals or their metabolites in body fluids or tissues, such as blood, urine, hairs, nails, and saliva. Blood and urine samples are the most widely accepted matrices for the biomonitoring of trace metal exposure, and there is less research on trace elements in saliva. Collection of saliva is simple and non-invasive, and saliva is much more accessible as compared to other sample mediums. In addition, dietary arsenic from seafood intake would be significant interference for the assessment of exposure to inorganic arsenic using urine arsenic levels as biological marker. Therefore, the purpose of this study was to determine the levels of trace elements in saliva and urine, including arsenic, barium, chromium, cobalt, cadmium, gallium, indium, lead, manganese, nickel, palladium, selenium, strontium, vanadium, and tungsten and arsenic species, i.e., arsenite (AsIII), arsenate (AsV), methylarsonic acid (MMA), dimethylarsinic acid (DMA) and arsenobetaine (AsB), and to evaluate the usability of saliva as biomonitoring medium. Mean while, fluctuation of salivary arsenic species levels after seafood intake was also examined to evaluate the effect of dietary arsenic intake on the inorganic metabolites in saliva. There are two parts in this study. In the first part, the levels of arsenic species and trace elements were determined in 34 saliva and urine samples, respectively, from 17 healthy volunteers and 17 patients from the Division of Nephrology in National Taiwan University Hospital. The levels of trace elements were determined by inductively coupled plasma mass spectrometry (ICP-MS), and the arsenic species were determined by high performance liquid chromatography with inductively coupled plasma mass spectrometry (HPLC-ICP-MS). There were positive correlations between salivary nickel level and urinary nickel level (rs = 0.503, p < 0.05), and between salivary tungsten level and urinary tungsten level (rs = 0.703, p < 0.05). There were significant differences in the saliva levels of arsenic, chromium, cobalt, lead, manganese, nickel, selenium, and vanadium among various age groups. The mean chromium level in saliva samples of volunteers with fixed orthodontic appliances or dentures was higher than that of volunteers without them. Higher salivary arsenate levels were observed in volunteers with kidney diseases. In the second part of this study, 16 volunteers were asked to eat about 100 g oyster or cuttlefish in one meal, and their saliva and urine samples were collected before and after the seafood intake for the determination of arsenic species levels. Results showed that the levels of arsenic in urine samples increased significantly after the intake of seafood, while there was no difference between arsenic levels in saliva before and after the seafood intake. In conclusion, tungsten in human saliva could be a useful biomarker to assess human exposure to tungsten. So were saliva arsenic species for the evaluation of inorganic arsenic exposure since they would not be affected by seafood arsenic intake.

Thesis deals with relationship between histomorphological and molecular-genetic findings of selected salivary gland tumors. Author, as a molecular-cytogeneticist mainly focused on detection of tumor-specific translocations of the salivary gland tumors which can serve as differential diagnostic markers. The thesis is composed as a commented files of authors own publications, and it is divided into four parts. First part deepens the knowledge of salivary adenoid cystic carcinoma. It was proved, that t(6;9)(q22-23;p23-24) resulting in fusion of transcription factors MYB-NFIB, or more rarely t(8;9) resulting in MYBL1-NFIB fusion represent robust differential diagnostic marker of adenoid cystic carcinoma. Further it was proved, that the 1p36 deletion can serve as an unfavorable prognostic indicator of adenoid cystic carcinoma, as the patients with 1p36 deletion had significantly lower survival. Second part summarizes new developments about mammary analogue secretory carcinoma (MASC), which was described by our group as a new salivary tumor entity characterized by translocation t(12;15)(p13;q25) resulting in ETV6-NTRK3 fusion. Another novel observation is a discovery of ETV6-RET fusion in a subset of MASC cases. Further, the first two MASCs of nasal mucosa origin have been described. Third part consists...