Fermentation and roasting of cocoa (Theobroma cacao) decrease levels of polyphenolic flavanol compounds. However, it is largely unknown how these changes in polyphenol levels caused by processing affect cocoa's anti-diabetic and anti-obesity bioactivities, such as inhibition of certain enzymes in the body. Polyphenol profiles, protein-binding abilities, presence of compounds termed oxidative polymers, and abilities to inhibit α-glucosidase, pancreatic α-amylase, lipase, and dipeptidyl peptidase-IV (DPP4) in vitro were compared between unfermented bean (UB), fermented bean (FB), unfermented liquor (UL), and fermented liquor (FL) cocoa extracts. Overall, there were significant decreases (p<0.05) in total polyphenols, flavanols, and anthocyanins between the two sets of unfermented and fermented cocoa extracts (CEs). All CEs effectively inhibited α-glucosidase (lowest IC50 = 90.0 ug/mL for UL) and moderately inhibited α-amylase (lowest IC50=183 ug/mL for FL), lipase (lowest IC25=65.5 ug/mL for FB), and DPP4 (lowest IC25=1585 ug/mL for FB) in dose-dependent manners. Fermentation and roasting of the samples affected inhibition of each enzyme differently (both processes enhanced α-amylase inhibition). Improved α-glucosidase and α-amylase inhibitions were correlated with presence of different classifications of oxidative polymers, suggesting that these compounds could be contributing to the bioactivities observed. Some α-glucosidase inhibition might be due to non-specific protein-binding. Improved DPP4 inhibition was strongly correlated to increased CE degree of polymerization. In conclusion, potential enzyme inhibition activities of cocoa were not necessarily negatively affected by the large polyphenol losses that occur during fermentation and roasting. Additionally, it is possible that more complex compounds could be present in cocoa that contribute to its potential anti-diabetic and anti-obesity bioactivities. / Master of Science in Life Sciences

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal. / L'amylose réticulé (AR), obtenu suite à la reticulation de l'ami don à haute teneur en amylose par traitement à l'épichlorhydrine, a été introduit au début des années 1990 comme excipient pour la libération contrôlée de médicaments. Divers taux de reticulation de l'amylose peuvent être obtenus en variant le rapport épichlorhydrine / amylose. Les cinétiques de libération de médicaments à partir de comprimés d'AR, en fonction du taux de reticulation du polymère, de la force de compression et de la charge en principe actif ont été étudiés. L'analyse cinétique de la liberation ainsi que l'absence d'une transition polymérique de l'état vitreux à caoutchouteux suggéraient l'implication d'un mécanisme de libération autre que la seule diffusion du principe actif. Une hypothèse d'un mécanisme de libération reposant sur la pénétration de l'eau dans le comprimé provoquant le remplacement des liens amylose-amylose par de nouveaux liens eau-amylose capables de freiner la pénétration de l'eau et la diffusion du principe actif avait été émise. Ce travail constitue la suite logique du développement et de la caractérisation physico-chimique de l'AR utilisé comme excipient pour la préparation de formes à liberation contrôlée de médicaments. La libération de principes actifs peu solubles en milieu aqueux à partir d'une matrice d'AR peut parfois être trop lente et s'effectuer sur plusieurs jours. Pour ces médicaments, la vitesse de libération peut être accélérée en ajoutant une quantité déterminée d'a-amylase dans le mélange pulvérulent d'AR et de principe actif, avant d'effectuer la compression. Une augmentation du contenu en a-amylase dans les comprimés provoque une augmentation marquée de la vitesse de libération et une augmentation de la linéarité des profils de libération, alors que de fortes modifications de la concentration d'a-amylase dans le milieu de dissolution présentent moins d'effet. Pour des concentrations élevées d'a-amylase, l'hydrolyse enzymatique devient le processus prédominant du contrôle de la libération du principe actif. Les profils de libération témoignent de l'influence de deux mécanismes qui régissent le contrôle de libération: i) un gonflement du comprimé accompagné par une diffusion du principe actif et ii) une hydrolyse enzymatique provoquant une érosion du comprimé et une accélération du taux de libération. En-dehors de ses aptitudes pour contrôler la libération de principes actifs, l'AR possède des propriétés liantes et peut aussi devenir un agent de désagrégation. La dureté augmente avec le degré de reticulation jusqu'à un maximum de dureté pour le degré AR-15. Une plus grande proportion d'AR ainsi qu'une augmentation de la force de compression ont pour conséquence d'augmenter la dureté et de diminuer la friabilité des comprimés. La présence de 0.2 % de stéarate de magnésium dans les comprimés contenant de l'AR ne semble pas avoir d'effet sur la dureté et la friabilité. Une étude comparative des propriétés liantes de l'amylose réticulé (AR-8) et de l'amylopectine réticulée à 8 % semble démontrer qu'une augmentation de la teneur en amylose conduit directement à une augmentation de la dureté des comprimés. Les résultats des tests de désagrégation se sont révélés satisfaisants lorsque l'AR 15 est comparé à l'Avicel PH-102®. La vitesse d'absorption d'eau des comprimés augmente lorsque le taux de reticulation de l'amylose augmente suggérant que les propriétés de désagrégation de l'AR (taux de reticulation supérieur à 15 %) soient reliées à une pénétration rapide de l'eau dans les comprimés. La caractérisation de l'AR par analyse d'image a permis de démontrer qu'un gel se forme à la surface des comprimés à base d'amylose réticulé en moins de 10 minutes suivant l'immersion des comprimés dans l'eau. La formation du réseau de gel de la surface vers le centre du comprimé s'effectue progressivement pendant plusieurs heures jusqu'à ce qu'un équilibre de gonflement soit atteint (environ 72 heures). Quelque soit le taux de reticulation, les spectres RMN des comprimés d'amylose réticulé s'apparentent au spectre Va de l'amidon. Suivant l'immersion dans l'eau des comprimés à base d'amylose ayant un faible taux de reticulation, on assiste à la naissance progressive d'un spectre décrivant la forme cristalline de type B de l'amylose caractérisée par la formation de doubles hélices. La gélatinisation, la reticulation et le traitement de l'amidon à haute teneiu- en amylose modifient profondément l'organisation granulaire de l'amidon natif, comme en témoignent les profils de rayons X. Cette modification s'accentue avec l'augmentation du degré de reticulation suggérant que l'hélice simple de l'amylose subit une déformation tant et si bien que la structure devient complètement déformée pour des taux de reticulation très élevés. Les résultats obtenus en FT-IR sont en accord avec les résultats de diffraction de rayons X démontrant que pour les degrés faibles de reticulation, l'association intra- et inter-chaînes d'amylose est favorisée. L'ensemble des études de caractérisation par gonflement, par analyse d'image, par diffraction de rayons X, par FT-IR et par RMN démontrent que la propriété d'excipient pour la libération contrôlée vient, vraisemblablement,de l'habileté de l'amylose de former des gels compacts qui ne s'érodent pas. La gélification s'effectue suite à l'immersion des comprimés dans l'eau et à la transformation de la partie amorphe de l'amylose en une structure thermodynamiquement plus stable constituée d'hélices d'amylose aimelées et arrangées dans une superstructure de type B. La reticulation modérée de l'amidon à haute teneur en amylose implique aussi le greffage des molécules d'amylopectine au sein des longues sections de chaînes d'amylose permettant ainsi l'obtention d'un gel plus souple et élastique, tout en demeurant relativement résistant aux enzymes amylolytiques. L'augmentation excessive du taux de reticulation conduit à la déformation des hélices d'amylose, ce qui a comme conséquence l'inhibition de la réassociation de l'amylose se traduisant par l'augmentation des vitesses de libération des principes actifs. Les temps de libération sont maximaux pour les produits ayant un taux de reticulation modéré ; dans ce cas, les forces de réassociation des chaînes d'amylose seraient parfaitement équilibrées par la souplesse et élasticité du gel résultant de la reticulation impliquant les chaînes d'amylose et d'amylopectine.

Neste trabalho, o gene da &#945;-amilase de Bacillus subtilis foi clonado em S. cerevisiae, por &#948;-integração. Foram construídos dois vetores: 1) o gene truncado da &#945;-amilase de B. subtilis (amyEt) com a sequência sinal própria, sob regulação do promotor e terminador ADH1 (&#948;AmyEt&#948;); 2) o gene da &#945;-amilase completo de B. subtilis (amyE), com a sequência sinal MF&#945; de S. cerevisiae, sob a regulação do promotor e terminador PGK (&#948;AmyE&#948;). Esses vetores foram empregados na transformação genética de linhagens S. cerevisiae, em co-transformação com o plasmídeo pAJ50, que permite a seleção positiva. Os clones transformantes cultivados em meio YPDA (0,5% amido) produziram halos de amilolise de diferentes tamanhos. Os que receberam o gene da &#945;-amilase completo sob a regulação de PGK (&#948;AmyE&#948;) apresentaram os melhores resultados para a hidrólise de amido. Nenhum clone teve o crescimento prejudicado em relação à linhagem controle selvagem. Estes resultados indicam que os novos vetores apresentam bom potencial para emprego na construção de linhagens industriais de S. cerevisiae. / At the present work, the &#945;-amylase gene from Bacillus subtilis was cloned in S. cerevisiae by &#948;-integration. We constructed two vectors: 1) a truncated &#945;-amylase gene of B. subtilis (amyEt) with its own signal sequence, under the regulation of the ADH1 promoter and terminator of S. cerevisiae (&#948;AmyEt&#948;), 2) a complete &#945;-amylase gene of B. subtilis (amyE), with the signal sequence of MF&#945; of S. cerevisiae under the regulation of PGK promoter and terminator (&#948;AmyE&#948;). These vectors were used to genetic transformation in S. cerevisiae strains, in co-transformation with pAJ50 plasmid , which allows positive selection. The transformant clones grown in YPDA (0.5% starch) produced amilolise halos of different sizes. Those which received the complete -amylase gene under the regulation of PGK (&#948;AmyE&#948;) showed the best results for starch hydrolysis. None of the clones had its growth capacity altered when compared to the wild type. These results indicate that these vectors have a good potential to be employed in industrial strains transformation of S. cerevisiae.

L’homéostasie buccale conditionne fortement les perceptions gustatives ; elle repose sur un épithélium buccal sain et le bon fonctionnement des glandes salivaires, qui sont finement régulés par hormones sexuelles. Le but de cette thèse était de savoir si une exposition orale en bisphénol A, un migrant d’emballage alimentaire et de composites dentaires, et une alimentation riche en phyto-œstrogènes (soja) pouvaient modifier l’homéostasie buccale. Deux expérimentations ont été conduites : une étude dose-effet du BPA (5µg à 12,5 mg/kj/j) chez le rat adulte, et une étude d’interaction d’un régime riche en soja sur les effets du BPA. Sur la base de tests gustatifs et d’une approche histologique et moléculaire (qPCR-TR), la première étude identifie pour la première fois une action du BPA sur la sècheresse buccale. Nous avons constaté que le BPA était responsable d’une moindre consommation d’eau (p<0.01), d’une préférence augmenté au sel (p<0.05) et diminué au sucre (p<0.05), et d’une altération des sécrétions salivaires. Une réversibilité partielle des effets à l’arrêt du traitement. A contrario un régime riche en phyto-œstrogène augmente la prise d’eau (p<10-6) et diminue la préférence au sel (p<0.05) par rapport à un régime semi-synthétique, et peut s’opposer aux effets du BPA. Ces études montrent que BPA et phyto-œstrogènes exercent des effets œstrogéniques agonistes ou antagonistes en fonction de la cible biologique considérée, et qu’un régime à base de soja peut gommer la plupart des effets observés / Oral homeostasis strongly influences taste perceptions. It depends on a healthy oral epithelium and salivary gland secretions, which are both regulated by sex hormones. The aim of this thesis was to identify the effect of an oral exposure to Bisphenol A, a food packaging and dental sealer component, and of a soy-diet containing phytoestrogens on oral homeostasisTwo experiments were conducted in adult rats: a dose-response study of BPA (5μg 12.5 mg / kj / day), and a study about the impact of a soy-diet on the BPA disrupting effects. By using gustation choice tests, and histological and qPCR-TR molecular approach, we identify for the first time an action of BPA on dry mouth. We found that BPA reduced water consumption (p <0.01), increased salt intake (p <0.05) and decreased sugar intake (p <0.05), and also modulated salivary secretions. These effects were partially reversed by stopping oral exposure. In contrast, the soy-diet increased water intake (p <10-6) and decreased in salt preference (p <0.05) by comparing to the semi-synthetic diet did, and may correct the effects of BPA.These studies show that BPA and phytoestrogens exert agonist or antagonist estrogenic effects depending on the biological target, and that a soy-based diet can erase most of the observed BPA effects

L’amertume fait partie intégrante de notre alimentation. Elle est par exemple fortement représentée dans certaines boissons (ex: café) ou dans certains légumes tels les crucifères. Néanmoins, la perception de l’amertume varie entre les individus et certains aliments considérés comme bénéfiques pour la santé peuvent être rejetés en raison de leur goût amer. Des facteurs génétiques (ex : polymorphisme génétique des récepteurs du goût amer) ou environnementaux (ex : âge, prise de médicaments) expliquent en partie les variations interindividuelles dans la perception de l’amertume. Cependant, d’autres facteurs péri-récepteurs pourraient intervenir, notamment la composition salivaire. Afin d’investiguer dans un premier temps le lien existant entre le protéome salivaire propre à un individu et sa sensibilité à l’amertume, le seuil de détection du goût amer de la caféine a été mesuré sur 29 hommes sains. Leur salive au repos a été étudiée par électrophorèse mono- et bidimensionnelle. L’analyse par électrophorèse bidimensionnelle de la salive au repos des 6 sujets les plus sensibles et 6 les sujets les moins sensibles à la caféine a permis la détection de 255 spots, dont 26 étaient significativement différents entre hyper- et hyposensibles. L’identification de ces 26 spots a révélé la surexpression de fragments d’alpha amylase, de fragments d’albumine sérique, et de sous-unités alpha de l’immunoglobuline A ainsi que la sous-expression de cystatine SN chez les hypersensibles. Ce dernier résultat a été confirmé par Western Blot. Ceci a permis de formuler une hypothèse sur le rôle de la protéolyse en bouche sur la sensibilité à l’amertume. Dans un deuxième temps et afin d’étudier l’effet des molécules amères sur la composition salivaire, une étude in vitro a été menée sur la lignée cellulaire de glandes salivaires humaines HSG différenciées en acini ou non. Après une mise au point des conditions de différenciation (culture dite en 3D), la cystatine SN a été détectée dans les cellules HSG par Western blot après traitement des cellules à la caféine, à la quinine, et à l’urée. Après traitement à la caféine à 5, 50 ou 100µM, une quantification par ELISA a mis en évidence que la cystatine SN était toujours plus abondante dans les cellules HSG différenciées que dans les cellules non-différenciées. Spécifiquement dans les cellules différenciées, l’exposition à la caféine induisait une sur-expression de cystatine SN, la teneur maximale en cystatine SN étant observée avec la caféine à 50 µM. La présence de cystatine SN a également été détectée dans les milieux de culture / Bitterness is present in every day beverages (e.g. coffee) and foods (e.g. vegetables such as cruciferous plants). However, bitterness is perceived differently among individuals and some foods considered as healthy may be rejected due to their bitter taste. Several genetic (eg. genetic polymorphism of bitter taste receptors) or environmental (eg. age, medications) factors partly explain the interindividual variability in bitterness perception. However, other peri-receptor factors may intervene, in particular salivary composition. First, in order to investigate the link between salivary proteome and sensitivity to bitterness, the detection threshold to the bitter taste of caffeine was measured in 29 male healthy subjects. Their resting saliva was studied by one- and two-dimensional electrophoresis. Two-dimensional electrophoresis revealed that 26 out of 255 spots were significantly different between the 6 hypersensitive and 6 hyposensitive subjects to the bitter taste of caffeine. Identification of the 26 spots revealed an overexpression of amylase-, serum albumin-, and immunoglobulin A fragments, and an underexpression of cystatin SN in hypersensitive subjects. The latter finding was confirmed by Western blotting. These results have led to formulate an hypothesis on the role of in-mouth proteolysis in bitterness perception. Second, in order to study the effect of bitter molecules on salivary composition, an in vitro study was performed on undifferentiated and differentiated human salivary cell line HSG. After setting the experimental conditions for HSG cell differentiation (culture in 3D conditions), cystatin SN was detected in HSG cells by Western blot after treatment with caffeine, quinine, and urea. After cell exposure with caffeine at 5, 50 and 100 µM, quantification by ELISA demonstrated that cystatin SN was always more abundant in differentiated vs undifferentiated HSG cells. Specifically in differentiated cells, caffeine exposure resulted in over-expression of cystatin SN, 50µM inducing the highest effect. Cystatin SN was also detected in culture media of the HSG cells

Neste trabalho, o gene da &#945;-amilase de Bacillus subtilis foi clonado em S. cerevisiae, por &#948;-integração. Foram construídos dois vetores: 1) o gene truncado da &#945;-amilase de B. subtilis (amyEt) com a sequência sinal própria, sob regulação do promotor e terminador ADH1 (&#948;AmyEt&#948;); 2) o gene da &#945;-amilase completo de B. subtilis (amyE), com a sequência sinal MF&#945; de S. cerevisiae, sob a regulação do promotor e terminador PGK (&#948;AmyE&#948;). Esses vetores foram empregados na transformação genética de linhagens S. cerevisiae, em co-transformação com o plasmídeo pAJ50, que permite a seleção positiva. Os clones transformantes cultivados em meio YPDA (0,5% amido) produziram halos de amilolise de diferentes tamanhos. Os que receberam o gene da &#945;-amilase completo sob a regulação de PGK (&#948;AmyE&#948;) apresentaram os melhores resultados para a hidrólise de amido. Nenhum clone teve o crescimento prejudicado em relação à linhagem controle selvagem. Estes resultados indicam que os novos vetores apresentam bom potencial para emprego na construção de linhagens industriais de S. cerevisiae. / At the present work, the &#945;-amylase gene from Bacillus subtilis was cloned in S. cerevisiae by &#948;-integration. We constructed two vectors: 1) a truncated &#945;-amylase gene of B. subtilis (amyEt) with its own signal sequence, under the regulation of the ADH1 promoter and terminator of S. cerevisiae (&#948;AmyEt&#948;), 2) a complete &#945;-amylase gene of B. subtilis (amyE), with the signal sequence of MF&#945; of S. cerevisiae under the regulation of PGK promoter and terminator (&#948;AmyE&#948;). These vectors were used to genetic transformation in S. cerevisiae strains, in co-transformation with pAJ50 plasmid , which allows positive selection. The transformant clones grown in YPDA (0.5% starch) produced amilolise halos of different sizes. Those which received the complete -amylase gene under the regulation of PGK (&#948;AmyE&#948;) showed the best results for starch hydrolysis. None of the clones had its growth capacity altered when compared to the wild type. These results indicate that these vectors have a good potential to be employed in industrial strains transformation of S. cerevisiae.

Summary: Salivary alpha-amylase: More than an enzyme - Investigating confounders of stress-induced and basal amylase activity (Dipl.-Psych. Jana Strahler) The hypothalamus-pituitary-adrenal (HPA) axis and the autonomic nervous system (ANS) are two of the major systems playing a role in the adaptation of organisms to developmental changes that threaten homeostasis. The HPA system involves the secretion of glucocorticoids, including cortisol, into the circulatory system. Numerous studies have been published that introduced salivary cortisol to assess HPA axis activity and therefore strengthens its role as an easy obtainable biomarker in stress research that can be monitored easily and frequently. Recent findings suggest a possible surrogate marker of autonomic activity due to autonomic innervation of salivary glands: salivary alpha-amylase (sAA). Up to date, additional methodological research is needed for a better understanding of the advantages and disadvantages of sAA activity in comparison to already established markers of ANS activity. The aim of the present thesis is to further our knowledge of confounders of sAA activity under basal and acute stress conditions and to strengthen the validity of this enzyme as an easy obtainable alternative for ANS testing. After introducing classical and modern stress concepts and stress system physiology (chapter 2), the reader is acquainted with anatomical basics of salivary gland innervation and secretion of salivary proteins, including sAA, due to autonomic innervation (chapter 3 and 4). Afterwards, a more nuanced review of methodological considerations of sAA determination shows gaps of knowledge concerning its usefulness as a marker of ANS activity (chapter 5). Given the fact that the integration of sAA into developmental and aging research is a relative recent phenomenon, several issues have to be addressed before a final conclusion could be drawn. Therefore, we conducted a series of studies incorporating these considerations regarding behavioral correlates of inter- and intraindividual differences in sAA activity with a special emphasis on older adults. Chapter 7 deals with sAA activity under psychological stress conditions in different age groups. Since vulnerability to disease and disease prevalence patterns change with age, it is important to investigate stress reactivity of people in different age groups. We therefore investigated children between 6 and 10 years, because childhood is a sensitive period of growth and development, and thus plays an important role for later life health. Young adults were included to represent the most studied human age group as a reference. Older adults between 59 and 61 years were investigated, because at this age the course is set for the further development of a person’s health in later life, and because autonomic stress responses in older age might be important determinants of cardiovascular and inflammatory aging. Our goal is to test for associations of sAA with more established stress system markers, i.e., salivary cortisol as outcome measurement of HPA reactivity, heart rate (HR) and heart rate variability (HRV) as markers for autonomic reactivity, and to directly compare these responses between different age groups across the life span. Secretion of sAA and cortisol was repeatedly assessed in 62 children, 78 young adults, and 74 older adults after exposure to a standardized psychosocial stressor, the Trier Social Stress Test. In addition, cardiovascular activity was measured in both adult groups. Older adults showed attenuated sAA, HR, and HRV responses. Furthermore, we found higher sAA but lower cortisol at baseline as well as lower sAA and cortisol responses in children. Age by sex interactions were observed only for cortisol with higher responses in older male participants. No associations between the parameters were found. Results in children and young adults confirm previous results. Overall, findings implicate sAA as an alternative or additional autonomic stress marker throughout the life span, with marked and rapid responsiveness to stress in three relevant age groups. The impact of age and chronic stress on basal sAA activity is the center of interest in chapter 8. We therefore assessed diurnal profiles of sAA and salivary cortisol in 27 younger and 31 older competitive ballroom dancers as well as 26 younger and 33 older age- and sex-matched controls. According to the Allostatic Load concept, repeated, non-habituating responses to social-evaluative conditions, which characterize the lives of competitive ballroom dancers, should be associated with stress system dysregulations. Furthermore, we expect to see an increased sympathetic drive associated higher overall alpha-amylase activity in older adults. Analyses revealed an elevated daily overall output of sAA in older adults while there was no effect of age on mean cortisol levels. Alterations of diurnal rhythms were only seen in younger male dancers showing a flattened diurnal profile of sAA and younger dancers and female older dancers showing a blunted diurnal rhythmicity of cortisol. Furthermore, we found a negative correlation between summary indices of basal sAA and the amount of physical activity. In conclusion, higher overall output of sAA in older adults was in line with the phenomenon of a “sympathetic overdrive” with increasing age. Furthermore, a lower output of sAA in people who are more physical active was in line with the hypothesis of an exercise-induced decrease of sympathetic activity. Taken together, results of chapter 7 and 8 show a clear impact of age on sAA activity, either under acute stress or basal conditions. One problem when integrating sAA into developmental and aging research is the use of adrenergic agonists and antagonists what is very common in older adults, i.e. antihypertensive drugs (AD). As well, the previously shown sympathetic overactivity that occurs with normal aging is associated with higher blood pressure (BP). Therefore, chapter 9 deals with a possible impact of high BP and AD on diurnal sAA activity in 79 older adults (33 normotensive adults, 16 medicated vs. 45 hypertensive adults, 34 medicated). Results showed a pronounced rhythm of sAA in all groups. Diurnal profiles differed significantly between men and women with men lacking the typical decrease of sAA in the morning and showing more pronounced alterations throughout the day. An effect of AD on sAA profiles and area under the curve values indicates that subjects not using AD´s show a heightened diurnal profile and a higher total output of sAA. Descriptively, this was also true for hypertensive older adults. Hypertensive subjects and those not using AD showed the highest diurnal output of sAA and the steepest slope. In sum, our results show an impact of antihypertensive medication and a difference between normotensive and hypertensive subjects on characteristics of diurnal sAA activity. Hence, findings are of particular interest in research using sAA as a prognostic indicator of pathological states and processes. Given the fact that hypertension was also shown to be associated with substantial changes of transmitters within the suprachiasmatic nucleus (SCN) - the “biological clock” that receives photic input from retinal glands via the retinohypothalamic pathway - and an altered output from the SCN to the sympathetic nervous system, we broaden the idea of a possible effect of different lighting conditions on morning sAA profiles in chapter 10. In a counterbalanced within-subjects design six men and 16 women of different ages collected sAA morning profiles on two consecutive days with leaving their shutters closed on the one day (= dark) and open their shutters on the other day (= bright). We were able to replicate earlier findings of light-induced changes of salivary cortisol with higher responses during the bright condition. On either day, women showed larger cortisol increases than men. Despite multisynaptic autonomic connections arising from the SCN projecting to multiple organs of the body, we could not find an effect of sunlight on sAA morning profiles. Evidence for circadian clock gene expression in human oral mucosa might account for this result and indicates that peripheral oscillators may act more like integrators of multiple different time cues, e.g. light, food intake, instead of a “master” oscillator (SCN). Results of chapter 7 to 10 provide clear evidence that sAA is heightened in states of autonomic arousal, i.e. stress, aging and hypertension, and that its circadian rhythmicity seems to be regulated rather integrative than directly via efferent input from hypothalamic SCN neurons. In chapter 11 this thesis tries to approach one central question: What is the biological meaning of the findings made? According to this enzyme´s anti-bacterial and digestive action short term changes might not have a biological meaning itself but rather reflect just a small part of multiple coordinated body responses to stressful stimuli. While the sympathetic branch of the ANS mainly stimulates protein secretion, the parasympathetic branch stimulates saliva flow. Acute stress responses might therefore be interpreted as reflecting predominant sympathetic activity together with parasympathetic withdrawal. The same mechanism could also be suitable for the finding of higher diurnal levels of sAA in older adults or hypertensive subjects reflecting a higher peripheral sympathetic tone in these groups. Diurnal profiles of sAA itself may reflect circadian changes in autonomic balance. Circadian rhythms are of great advantage since they enable individuals to anticipate. This pre-adaptation enables the individual to cope with upcoming demands and challenges. Our finding of a relationship between sAA and salivary cortisol what strengthens the relevance of glucocorticoids that were previously shown to be able to phase shift circadian rhythms in cells and tissue. Within a food-related context there is evidence that decreasing levels of sAA in the morning could reflect increases of feeling hungry since sAA systematically increases during food consumption and with the subjective state of satiety. So far, much more research is needed to identify underlying physiological mechanisms of circadian sAA rhythmicity. Taking the next step, future studies will have to focus on the integration of sAA assessment into longitudinal studies and different disease states to prove its applicability as a marker of sympathetic neural functioning in the genesis and prognosis of disease.:Table of Contents 1. Introduction 1 2. Stress 3 2.1. Stress concepts 3 2.1.1. Traditional concepts of stress 3 2.1.2. Allostasis and Allostatic Load 4 2.2. Stress system physiology 6 2.2.1. The hypothalamic-pituitary-adrenal (HPA) axis 6 2.2.1.1. Physiology 6 2.2.1.2. HPA axis activity indicators 6 2.2.2. The autonomic nervous system (ANS) 7 2.2.2.1. Physiology 7 2.2.2.2. ANS activity indicators 8 2.2.3. Relationships between stress systems 10 3. Saliva and salivary glands 11 3.1. Physiology 11 3.1.1. Anatomy, origin, and composition 11 3.1.2. Innervation 12 3.1.3. Salivary gland physiology with aging 13 3.2. Saliva and salivary flow 13 3.3. Protein secretion 14 4. Alpha-amylase in saliva 15 4.1. Chemical characteristics 15 4.2. Secretion of alpha-amylase 15 4.3. Diagnostic value of alpha-amylase 16 5. Methodological considerations of alpha-amylase determination 17 5.1. Collection methods and preparation 17 5.1.1. Saliva collection 17 5.1.2. Impact of flow rate 17 5.1.3. Impact of pH-value 18 5.2. Biochemical determination 18 5.3. Interindividual differences in sAA activity 19 5.3.1. Basal activity 20 5.3.2. Acute responses 20 5.3.3. Age effects 21 5.3.3.1. Basal amylase activity 21 5.3.3.2. Stress-induced amylase activity 21 5.3.4. Sex differences 22 5.3.4.1. Basal amylase activity 22 5.3.4.2. Stress-induced amylase activity 23 5.3.5. Modulating factors influencing amylase (re-)activity 24 5.3.5.1. Impact of smoking 24 5.3.5.2. Impact of alcohol 25 5.3.5.3. Impact of caffeine 25 5.3.5.4. Impact of high body fat and obesity 26 5.3.5.5. Impact of food intake 26 5.3.5.6. Impact of physical exercise 27 5.3.5.7. Impact of somatic and psychiatric diseases 27 5.3.5.8. Impact of medical drugs 29 5.3.5.9. Impact of sunlight on diurnal amylase 29 6. Aims and outline of the present work 31 7. Salivary alpha-amylase stress reactivity across different age groups 32 7.1. Introduction 32 7.2. Methods 35 7.2.1. Participants 35 7.2.2. Study Protocol 35 7.2.3. Measures 36 7.2.3.1. Saliva sampling 36 7.2.3.2. Heart rate and heart rate variability 36 7.2.3.3. Biochemical analyses 37 7.2.3.4. Psychometrical analyses 37 7.2.4. Statistical analyses 38 7.3. Results 38 7.3.1. Sample characteristic 38 7.3.2. Subjective stress response 39 7.3.3. Physiological stress response 39 7.3.3.1. Salivary alpha-amylase 39 7.3.3.2. Salivary cortisol 40 7.3.3.3. Heart rate 42 7.3.3.4. Heart rate variability 43 7.3.3.5. Determinants of the salivary alpha-amylase stress response 45 7.4. Discussion 45 7.5. Conclusion 48 8. Aging diurnal rhythms and chronic stress: Distinct alteration of diurnal rhythmicity of salivary alpha-amylase and cortisol 49 8.1. Introduction 49 8.2. Methods 52 8.2.1. Participants 52 8.2.2. Study protocol 53 8.2.3. Measures 53 8.2.3.1. Saliva sampling 53 8.2.3.2. Biochemical parameters 54 8.2.3.3. Psychological parameters 54 8.2.4. Statistical analyses 54 8.2.4.1. Preliminary analyses 54 8.2.4.2. Diurnal course of salivary alpha-amylase 55 8.3. Results 56 8.3.1. Sample characteristic 56 8.3.2. Preliminary analyses: impact of oral contraceptives, blood pressure, and lipid lowering medication on diurnal profiles 56 8.3.3. Diurnal course of salivary alpha-amylase 57 8.3.3.1. Salivary alpha-amylase over the day 57 8.3.3.2. Salivary alpha-amylase after awakening 58 8.3.4. Diurnal course of salivary cortisol 59 8.3.4.1. Salivary cortisol over the day 59 8.3.4.2. Salivary cortisol after awakening 60 8.3.5. Diurnal course of salivary biomarkers: associations and determinants 61 8.4. Discussion 62 8.5. Conclusion 65 9. Impact of blood pressure and antihypertensive drugs on diurnal alpha-amylase activity: A novel marker of sympathetic drive 67 9.1. Introduction 67 9.2. Methods 68 9.2.1. Participants 68 9.2.2. Study protocol 69 9.2.3. Measures 69 9.2.3.1. Saliva sampling 69 9.2.3.2. Biochemical parameters 69 9.2.3.3. Blood pressure assessment 70 9.2.4. Statistical analyses 70 9.3. Results 70 9.3.1. Participants 70 9.3.2. Salivary alpha-amylase 71 9.3.2.1. Salivary alpha-amylase over the day 71 9.3.2.2. Salivary alpha-amylase after awakening 74 9.4. Discussion 75 9.5. Perspectives 76 10. Light affects morning salivary cortisol, but not salivary alpha-amylase 77 10.1. Introduction 77 10.2 Methods 79 10.2.1. Participants 79 10.2.2. Study protocol 80 10.2.3. Measures 80 10.2.3.1. Saliva sampling 80 10.2.3.2. Biochemical parameters 81 10.2.4. Statistical analyses 81 10.3. Results 82 10.3.1. Sociodemographics 82 10.3.2. Salivary alpha-amylase 82 10.3.3. Salivary cortisol 84 10.3.4. Associations between biochemical parameters 85 10.4. Discussion 86 10.5. Conclusion 89 11. General discussion 90 11.1. Summary of the results 90 11.1.1. Salivary alpha-amylase stress reactivity across different age groups 91 11.1.2. Aging diurnal rhythms and chronic stress: Distinct alteration of diurnal rhythmicity of salivary alpha-amylase and cortisol 91 11.1.3. Impact of blood pressure and antihypertensive drugs on diurnal alpha-amylase activity: A novel marker of sympathetic drive 92 11.1.4. Light affects salivary morning cortisol, but not salivary alpha-amylase 93 11.2. Integration of main findings 93 11.3. Stress-induced amylase activity, basal rhythm, and its biological meaning 95 11.4. Methodological consequences 97 11.4.1. Circadian variation 97 11.4.2. Longitudinal variation 98 11.4.3. Short-term variation and stability 98 11.4.4. Long-term change 99 11.5. Outlook 100 12. References 102

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Among adolescents, the day-to-day functioning of the hypothalamo-pituitary-adrenal-axis (HPA-axis) and of the autonomic nervous system (ANS) and their relationships with stress, subjective health complaints and psychological factors such as self-esteem, studied in naturalistic settings, have been largely unexplored. This thesis aimed to investigate the diurnal activity of the HPA-axis (Studies I &amp; II) in terms of salivary cortisol and the ANS/SNS system (Study III) in terms of salivary alpha-amylase (sAA) in mid-adolescent girls and boys. Additionally, linkages between self-reported stress, self-esteem, recurrent pain and biomarkers were investigated. A further aim was to describe potential differences between girls and boys respectively. Study I showed that both girls and boys exhibited the typical diurnal cortisol profile with high levels in the morning that decreased throughout the day. Girls had higher total cortisol levels, while no differences emerged for measures of the cortisol increase. Study II showed no significant linkages between self-ratings of stress and cortisol. However, stress was associated with recurrent pain in girls. Study III showed that, for girls, both self-esteem and self-reported stress were related to morning levels of both cortisol and sAA, to the diurnal sAA output and to a conjoint measure of amylase over cortisol, AOC. To conclude, the findings suggest that both stress and self-esteem may be linked to different measures of ANS and HPA-axis activity, but also to measures of ANS and HPA-axis dysregulation, particularly among mid-adolescent girls. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: Manuscript.</p>

A mandioca (Manihot esculenta Crantz) é uma raiz originária e cultivada na América do Sul, com alta perecibilidade no período pós-colheita. Seus principais processos de deterioração envolvem reações enzimáticas, oxidativas e microbiológicas. Neste trabalho foram estudadas raízes de mandioca da variedade Zolhudinha catalogada pela EMBRAPA como IM-158, provenientes da região amazônica, que se destacam pela alta atividade amilolítica. Foram analisadas as condições físico-químicas junto com o isolamento e purificação da &#945;-amilase da raiz e a possível participação desta enzima no processo deteriorativo pós-colheita. Por ser uma variedade de mandioca não comercial, o tempo de cocção foi em média de 4,30 h, teor de umidade em tomo de 64 % e porcentagem de amido de cerca de 30 %. A atividade amilásica decai em 1/3 de sua intensidade no quinto dia pós-colheita, em contraponto a formação de açúcares redutores, cuja concentração aumenta cinco vezes. A purificação foi obtida com duas etapas cromatográficas, com DEAE-celulose e Sephacryl S-200, revelando duas isoenzimas de &#945;-amilase treze vezes mais purificadas, com recuperação protéica de 7,5 % e com pesos moleculares entre 14 e 19 kDa. / Cassava (Manihot esculenta Crantz) is a root from a native plant, cultivated in South América, that is hightly perish on the post-harvest time. The mechanisms of the root deterioration are due to enzymatic and oxidative reactions as well as the microbiological attack. In this work were studied roots of Zolhudinha variety, EMBRAPA - IM 158, cultivated in Amazonian area, which distinguishes from others varieties by its higher amylase activity. Physicochemical properties were analyzed during the post-harvest time, the purification of &#945;-amylase were performed to establish a possible involvement on the deteriorative process. As a non-commercial variety, the cooking time of the roots was 4.30 hours on average, with 64 % of water content and 30 % of starch. The amylase activity during the post-harvest decrease 1/3 from the original at the day 5, that matches with the reducing sugar in roots by increase of five times. The purification was achieved by two chromatography steps on DEAE-cellulose and Sephacryl S-200, providing two isozymes of &#945;-amylase, thirteen times more purified with a recovery of 7.5 % of the protein fraction, the estimated molecular weights were between 14 and 19 kDa.