It has been repeatedly shown that functional magnetic resonance imaging (fMRI) triggers distress and neuroendocrine response systems. Prior studies have revealed that sympathetic arousal increases, particularly at the beginning of the examination. Against this background it appears likely that those stress reactions during the scanning procedure may influence task performance and neural correlates. However, the question how sympathetic arousal elicited by the scanning procedure itself may act as a potential confounder of fMRI data remains unresolved today. Thirty-seven scanner naive healthy subjects performed a simple cued target detection task. Levels of salivary alpha amylase (sAA), as a biomarker for sympathetic activity, were assessed in samples obtained at several time points during the lab visit. SAA increased two times, immediately prior to scanning and at the end of the scanning procedure. Neural activation related to motor preparation and timing as well as task performance was positively correlated with the first increase. Furthermore, the first sAA increase was associated with task induced deactivation (TID) in frontal and parietal regions. However, these effects were restricted to the first part of the experiment. Consequently, this bias of scanner related sympathetic activation should be considered in future fMRI investigations. It is of particular importance for pharmacological investigations studying adrenergic agents and the comparison of groups with different stress vulnerabilities like patients and controls or adolescents and adults.

We often form intentions but have to postpone them until the appropriate situation for retrieval and execution has come, an ability also referred to as event-based prospective memory. After intention completion, our cognitive system has to deactivate no-more-relevant intention representations from memory to avoid interference with subsequent tasks. In everyday life, we frequently rely on these abilities also in stressful situations. Surprisingly, little is known about potential stress effects on these functions. Therefore, the present study aimed to examine the reliability of event-based prospective memory and of intention deactivation in conditions of acute psychosocial stress. To this aim, eighty-two participants underwent the Trier Social Stress Test, a standardized stress protocol, or a standardized control situation. Following this treatment, participants performed a computerized event-based prospective memory task with non-salient and focal prospective memory cues in order to assess prospective memory performance and deactivation of completed intentions. Although the stress group showed elevated levels of salivary cortisol as marker of a stress-related increase in hypothalamus-pituitary-adrenal axis activity throughout the cognitive testing period compared to the no-stress group, prospective memory performance and deactivation of completed intentions did not differ between groups. Findings indicate that cognitive control processes subserving intention retrieval and deactivation after completion may be mostly preserved even under conditions of acute stress.

Diss. (sammanfattning) Umeå : Umeå universitet, 2010.

Nous avons étudié le rôle de l'immunité innée de la peau lors de la transmission des Borrelia (agent infectieux de la borréliose de Lyme) par son vecteur, une tique dure du genre Ixodes. Nous avons montré que la salive de tique et la protéine salivaire Salp15 inhibent la réaction inflammatoire (production de chimiokines et de peptides antimicrobiens) des kératinocytes induite par Borrelia. Cet effet anti- « alarmine » de la salive de tique contribue probablement à créer un environnement cutané local favorable à la transmission de Borrelia. Nous avons montré que Borrelia induit également au niveau des fibroblastes cutanés la transcription de nombreux gènes proinflammatoires. Nous avons observé un effet toxique direct de la salive de tique sur les fibroblastes cutanés : cet effet dose-dépendant est de nature protéique mais non lié à la protéine Salp15. Ces résultats indiquent que les fibroblastes jouent un rôle important dans l’inflammation cutanée induite par Borrelia. / We studied the role of the skin innate immunity during the transmission of Borrelia (the infectious agent of Lyme borreliosis) by its vector, a hard tick belonging to the genus Ixodes. We showed that tick saliva and its protein Salp15 both inhibate Borrelia-induced inflammatory reaction of keratinocytes. The antialarmin effect of tick saliva ensure a favorable environment for Borrelia. We also showed that Borrelia induce a strong inflammatory response in dermal fibroblasts. We also demonstrate a dose-dependent lytic effect of tick salivary gland extracts on dermal fibroblasts and that this cytotoxic effect was of proteinaceous nature and not related to Salp15. These results indicate that dermal fibroblasts could be considered as central mediators in immune cell recruitment to the skin site of Borrelia invasion.

I characterized several potential glycoproteins in salivary gland extracts from unfed and partially fed females of ticks Ixodes ricinus using enzyme deglycosylation and lectin labeling. Affinity-based (chromatografic) analysis was applied for isolations of glycoproteins with specificity for GNA (mannose), HPA (N-acetylgalactosamine) and MAA II (sialic acid) lectins. GNA specific 120 kDa glycoprotein was isolated from partially fed females and is modified with N-linked glycans containing {$\alpha$}1,3-mannose. Mass spectrometry analyses confirmed the presence carboxypeptidase M in elution fraction gain with GNA affinity chromatography. GNA specific proteins were purified from unfed female salivary gland extracts. MS analyses identified them as proteins similar to arylsulfatase B and cytoskeletal Sojo protein. Proteins (85 and 56 kDa) isolated with HPA affinity chromatography were characterized as Trappin 12, which is a host protein. MAA II lectin was used for labelling and isolation of 100 kDa protein. N-terminal sequence of the MAA II specific protein predicted similarity with a host protein, Siglec 1. Fucose in salivary gland extract was detected with the labelling of AAA, AAL, UEA I and LTL lectins. Results showed that salivary gland extracts contain {$\alpha$}1,2-; {$\alpha$}1,3- and {$\alpha$}1,6- N-linked fucose and O-linked fucose probably as well. GNA specific proteins were detected in partially fed salivary glands acini type II and III using electron transmission microscopy. Fucose was detected on gut and salivary gland structures using fucose-specific lectin AAL.

La naissance est l’une des étapes les plus délicates à laquelle les nouveau-nés mammifères doivent faire face. Le nouveau-né doit opérer des changements physiologiques et comportementaux pour s’adapter à l’environnement aérien, et l’un des premiers défis est d’ingérer du colostrum et du lait. Il est surprenant que la nature des stimuli et les mécanismes impliqués dans le déclenchement de la tétée soient encore mal connus, alors que la survie du nouveau-né est conditionnée par le succès de la première tétée. Par conséquent, cette étude a pour but de comprendre comment un nouveau-né immature et inexpérimenté réussit à s’orienter vers une tétine, à la saisir et à la téter de façon efficace? Cette question générale est posée chez la souris en focalisant sur : i) la nature des substrats chimiques utilisés par les souriceaux pour atteindre les tétines maternelles ; ii) la variation de la puissance attractive de ces substrats au cours du développement ; et iii) l’implication des effets de l’expérience dans l’établissement des réponses adaptatives précoces. Premièrement, nos résultats mettent en lumière que les odeurs mammaires de femelles allaitantes induisent plus d’approches et de saisies de la tétine chez les souriceaux que celles émanant de femelles non allaitantes. Deuxièmement, les odeurs de liquide amniotique et de lait déclenchent la première saisie orale de la tétine chez des souriceaux à la naissance, alors que les odeurs de salives maternelle et infantile n’induisent ce comportement qu’après une brève expérience de tétée. Troisièmement, les souriceaux âgés de 0, 2 et 6 jours postnatals (P), ayant eu une expérience de tétée, affichent une attraction sélective envers des odeurs de laits collectés en début de lactation plutôt qu’en fin de lactation, alors que les souriceaux plus âgés P15 ne montrent aucune réponse sélective envers ces odeurs. En résumé, certains substrats biologiques présents sur les tétines de femelles allaitantes sont immédiatement attractifs après la naissance, tandis que d’autres ont besoin d’être appris pour être réactogènes. Par conséquent, la réponse initiale de recherche de la tétine chez le souriceau est contrôlée par des processus d’apprentissage postnatal. A ce stade, l’implication de l’apprentissage prénatal et de processus prédisposés n’a pu être prouvée, bien qu’elle ne soit pas exclue. Ces résultats montrent des capacités d’apprentissage sophistiquées chez le souriceau nouveau-né / Birth is one of the most delicate periods mammalian infants have to deal with. Newborns have then to adapt physiologically and behaviorally to the aerial environment, and one of their first challenges is to ingest colostrum and milk. It is paradoxical that the survival of pups is conditioned by the success of this first suckling, and that we have so little understanding of the stimuli that underlie and promote it. Thus, the present work aims to contribute to answer how immature and naïve newborns do manage to orient to a nipple, to grasp it, and to suckle efficiently? This general issue will be addressed in the mouse in focusing on: i) the nature of the chemical substrates that newborn mice use to reach nipples; ii) whether the attractive potency of these substrates changes as a function of development; and iii) whether exposure effects underly the establishment of early adaptive responses? The results highlight that mammary odors of lactating females are more behaviorally active for newly born pups than those of non-lactating females. Secondly, amniotic and milk odors provoke the first nipple grasping in newborns right at birth, while maternal and pup salivary odors induce this behavior after short sucking experience. Thirdly, younger pups on postnatal day (P) 0, 2 and 6 (with sucking experience), display a selective orientation toward the odor of milk collected during early-lactation rather than to the odor of late-lactation milk, whereas older pups P15 do not exhibit any selective attraction to these odors. To summarize, some of the biological substrates which are present on a nursing mouse nipples are attractive immediately after birth, while some others need to be postnatally learned to be active. Thus, the initial nipple search response of mouse pups is controlled by processes involving postnatal learning. At this stage, the involvement of prenatal learning and of predisposed processes that do not depend on previous exposure effect are not conclusive, although not excluded. These results show highly sophisticated learning abilities in a newborn mammal

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

Objectives: Dysregulation of the hypothalamus-pituitary-adrenal (HPA) axis has frequently been reported in multiple sclerosis (MS). So far, HPA axis function in MS has predominantly been studied under pharmacological stimulation which is associated with a series of methodological caveats. Knowledge of circadian cortisol patterns and cortisol awakening response (CAR) is still limited. Methods: A total of 77 MS patients (55 relapsing-remitting MS (RRMS)/22 secondary-progressive MS (SPMS)) as well as 34 healthy control (HC) subjects were enrolled. Diurnal cortisol release was assessed by repeated salivary cortisol sampling. Neurological disability was rated by the Kurtzke’s Expanded Disability Status Scale (EDSS). Depressive symptoms and perceived stress were assessed by self-report measures. Results: RRMS but not SPMS patients differed in circadian cortisol release from HC subjects. Differences in cortisol release were restricted to CAR. Treated and treatment naïve RRMS patients did not differ in CAR. In a RRMS follow-up cohort (nine months follow-up), RRMS patients with EDSS progression (≥0.5) expressed a significantly greater CAR compared to HC subjects. RRMS patients with a stable EDSS did not differ from HC subjects. Neither depressive symptoms nor perceived stress ratings were associated with CAR in RRMS patients. In a step-wise regression analysis, EDSS at baseline and CAR were predictive of EDSS at follow-up (R2 = 67%) for RRMS patients. Conclusions: Circadian cortisol release, in particular CAR, shows a course specific pattern with most pronounced release in RRMS. There is also some evidence for greater CAR in RRMS patients with EDSS progression. As a consequence, CAR might be of predictive value in terms of neurological disability in RRMS patients. The possible role of neuroendocrine-immune interactions in MS pathogenesis is further discussed.

It has been repeatedly shown that functional magnetic resonance imaging (fMRI) triggers distress and neuroendocrine response systems. Prior studies have revealed that sympathetic arousal increases, particularly at the beginning of the examination. Against this background it appears likely that those stress reactions during the scanning procedure may influence task performance and neural correlates. However, the question how sympathetic arousal elicited by the scanning procedure itself may act as a potential confounder of fMRI data remains unresolved today. Thirty-seven scanner naive healthy subjects performed a simple cued target detection task. Levels of salivary alpha amylase (sAA), as a biomarker for sympathetic activity, were assessed in samples obtained at several time points during the lab visit. SAA increased two times, immediately prior to scanning and at the end of the scanning procedure. Neural activation related to motor preparation and timing as well as task performance was positively correlated with the first increase. Furthermore, the first sAA increase was associated with task induced deactivation (TID) in frontal and parietal regions. However, these effects were restricted to the first part of the experiment. Consequently, this bias of scanner related sympathetic activation should be considered in future fMRI investigations. It is of particular importance for pharmacological investigations studying adrenergic agents and the comparison of groups with different stress vulnerabilities like patients and controls or adolescents and adults.

Transdisciplinary research in general, and stress research in particular, requires an efficient integration of methodological knowledge of all involved academic disciplines, in order to obtain conclusions of incremental value about the investigated constructs. From a psychologist’s point of view, biochemistry and quantitative neuroendocrinology are of particular importance for the investigation of endocrine stress systems (i.e., the HPA axis, and the SNS). Despite of their fundamental role for the adequate assessment of endocrine activity, both topics are rarely covered by conventional psychological curriculae. Consequently, the transfer of the respective knowledge has to rely on other, less efficient channels of scientific exchange. The present thesis sets out to contribute to this exchange, by highlighting methodological issues that are repeatedly encountered in research on stress-related endocrine activity, and providing solutions to these issues. As outlined within this thesis, modern stress research tends to fall short of an adequate quantification of the kinetics and dynamics of bioactive cortisol. Cortisol has gained considerable popularity during the last decades, as its bioactive fraction is supposed to be reliably determinable from saliva and is therefore the most conveniently obtainable marker of HPA activity. However, a substantial fraction of salivary cortisol is metabolized to its inactivated form cortisone by the enzyme 11β-HSD2 in the parotid glands, which is likely to restrict its utility. Although the commonly used antibody-based quantification methods (i.e. immunoassays) might “involuntarily” qualify this issue to some degree (due to their inherent cross-reactivity with matrix components that are structurally-related to cortisol; e.g., cortisone), they also cause differential within-immunoassay measurement bias: Salivary cortisone has (as compared to salivary cortisol) a substantially longer half-life, which leads to an overestimation of cortisol levels the more time has passed since the onset of the prior HPA secretory episode, and thus tends to distort any inference on the kinetics of bioactive cortisol. Furthermore, absolute cortisol levels also depend on the between-immunoassay variation of antibodies. Consequently, raw signal comparisons between laboratories and studies, which are favorable as compared to effect comparisons, can hardly be performed. This finding also highlights the need for the long-sought standardization of biochemical measurement procedures. The presumably only way to circumvent both issues is to rely on quantification of ultrafiltrated blood cortisol by mass-spectrometric methods. Being partly related to biochemical considerations with research on HPA activity, a second topic arises concerning the operationalization of the construct itself: In contrast to the simple outcome measures like averaged reaction times, inclined stress researchers can only indirectly infer on the sub-processes being involved in HPA activity from longitudinally sampled hormone concentrations. HPA activity can be quantified either by (a) discrete-time, or by (b) continuous-time models. Although the former is the most popular and more convenient approach (as indicated by the overly frequent encounter of ANOVAs and trapezoidal AUC calculations in the field of psychobiological stress research), most discrete time models form rather data-driven, descriptive approaches to quantify HPA activity, that assume the existence of some endocrine resting-state (i.e., a baseline) at the first sampling point and disregard any mechanistic hormonal change occurring in between all following sampling points. Even if one ignores the fact, that such properties are unlikely to pertain to endocrine systems in general, many generic discrete time models fail to account for the specific structure of endocrine data that results from biochemical hormone measurement, as well as from the dynamics of the investigated system. More precisely speaking, cortisol time series violate homoscedasticity, residual normality, and sphericity, which need to be present in order to enable (mixed effects) GLM-based analyses. Neglecting these prerequisites may lead to inference bias unless counter-measures are taken. Such counter-measures usually involve alteration of the scale of hormone concentrations via transformation techniques. As such, a fourth-root transformation of salivary cortisol (being determined by a widely used, commercially available immunoassay) is shown to yield the optimal tradeoff for generating homoscedasticity and residual normality simultaneously. Although the violation of sphericity could be partly accounted for by several correction techniques, many modern software packages for structural equation modeling (e.g., Mplus, OpenMX, Lavaan) also offer the opportunity to easily specify more appropriate moment structures via path notation and therefore to relax the modeling assumptions of GLM approaches to the analysis of longitudinal hormone data. Proceeding from this reasoning, this thesis illustrates how one can additionally incorporate hypotheses about HPA functioning, and thus model all relevant sub-processes that give rise to HPA kinetics and dynamics. The ALT modeling framework being advocated within this thesis, is shown to serve well for this purpose: ALT modeling can recover HPA activity parameters, which are directly interpretable within a physiological framework, that is, distinct growth factors representing the amount of secreted cortisol and velocity of cortisol elimination can serve to interpret HPA reactivity and regulation in a more unambiguous way, as compared to GLM effect measures. For illustration of these advantages on a content level, cortisol elimination after stress induction was found to be elevated as compared to its known pharmacokinetics. While the mechanism behind this effect requires further investigation, its detection would obviously have been more difficult upon application of conventional GLM methods. Further extension of the ALT framework allowed to address a methodological question, which had previously been dealt with by a mere rule of thumb; what’s the optimal threshold criterion, that enables a convenient but comparably accurate classification of individuals whose HPA axis is or is not activated upon encountering a stressful situation? While a rather arbitrarily chosen baseline-to-peak threshold of 2.5 nmol/L was commonly used to identify episodes of secretory HPA activity in time series of salivary cortisol concentrations, a reanalysis of a TSST meta- dataset by means of ALT mixture modeling suggested that this 2.5 nmol/L criterion is overly conservative with modern biochemical measurement tools and should be lowered according to the precision of the utilized assay (i.e., 1.5 nmol/L). In sum, parametric ALT modeling of endocrine activity can provide a convenient alternative to the commonly utilized GLM-based approaches that enables the inference on and quantification of distinct HPA components on a theoretical foundation, and thus to bridge the gap between discrete- and continuous-time modeling frameworks. The implementation of the outlined modeling approaches by the respective statistical syntaxes and practical guidelines being derived from the comparison of cortisol assays mentioned above, are provided in the appendix of the present thesis, which will hopefully help stress researchers to directly quantify the construct they actually intend to assess.:1. Introduction 2. The hypothalamus-pituitary-adrenal (HPA) axis 3. Induction and quantification of HPA activity 4. The pitfalls of SCC measurement 5. Creating normality and homoscedasticity: GLM-based analyses 6. Relaxing sphericity: moment structure analyses 7. General conclusion