Neuweltkameliden, Trächtigkeitsdiagnose, Hormone, Speichel, Milch, Urin Ziel der vorliegenden Arbeit war es, die trächtigkeitsassoziierten Hormone Progesteron (P4), Pregnanediol-Glucuronid (PdG), Östronsulfat (E1S) und Relaxin (RLN) in Spei-chel, Milch und Urin von tragenden und nicht tragenden Alpakas im Vergleich zur je-weiligen Blutkonzentration zu bestimmen, um ihre Eignung zur nicht invasiven Träch-tigkeitsdiagnostik zu untersuchen. Beprobt wurden, über einen Zeitraum von zwei Jahren, 36 Alpakastuten von sechs pri-vaten Züchtern in Sachsen jeweils vor der Bedeckung und in verschiedenen Stadien der Trächtigkeit (verifiziert durch eine transabdominale Ultraschalluntersuchung). Es wurden jeweils Serum-, Plasma-, Speichel-, Urin- und Milchproben gewonnen und die Hormonkonzentrationen mittels Enzymimmunoassay (EIA) bestimmt. Weiterhin wurden einige Milchproben in einem semiquantitativen Progesteron-Schnelltest für Rinder ein-gesetzt. P4-Konzentrationen steigen signifikant von Basalwerten beim nicht tragenden Tier von 0,35 ± 0,04 ng/ml auf 2,94 ± 0,11 ng/ml Plasma (bzw. von 0,26 ± 0,03 auf 2,87 ± 0,10 ng/ml Serum) bei tragenden Tieren an. Auch in Milch und im Urin tragender Alpakas sind signifikant höhere P4-Konzentrationen messbar: Sie steigen von basal 0,83 ± 0,06 ng/ml auf 4,09 ± 0,38 ng/ml Milch bzw. von 0,29 ± 0,04 ng P4/mg Krea auf 0,60 ± 0,06 ng P4/mg Krea im Urin. Die Urin-Konzentrationen von PdG sind signifikant höher bei graviden (152,73 ± 17,37 ng PdG/mg Krea) als bei ingraviden Alpakas (26,70 ± 2,80 ng PdG/mg Krea). Im Speichel sind weder von P4 noch von PdG Konzentrationsunterschiede zwischen den beiden Gruppen nachweisbar. Der P4-Schnelltest erkannte 28 von 31 Milchproben tragender Tiere richtig als tragend, was einem Prozentsatz von 90 % entspricht. Dage-gen wurden 22 von 32 Proben nicht tragender Tiere als nicht tragend identifiziert (69 %), wobei von den falsch positiven Milchproben jedoch 70% auch mit dem labor-gebundenen EIA falsch positive Ergebnisse lieferten. Während Blutkonzentrationen von RLN signifikant nach dem zweiten Trächtigkeitsmo-nat von basal 1,65 ± 0,56 ng/ml auf 11,69 ± 2,31 ng/ml (Plasma) bzw. von 0,95 ± 0,30 ng/ml auf 16,23 ± 3,05 ng/ml (Serum) ansteigen, sind keine Unterschiede in Milch, Speichel und Urin zwischen tragenden und nicht tragenden Tieren nachweisbar. Konzentrationen von E1S steigen erst im letzten Trächtigkeitsmonat signifikant an: Blutwerte steigen von basal 0,59 ± 0,07 ng/ml auf 3,43 ±0,55 ng/ml (Plasma) bzw. 0,32 ± 0,02 ng/ml auf 2,16 ± 0,43 ng/ml (Serum) und Urinwerte von basal 6,14 ± 0,53 ng E1S/mg Krea auf 104,03 ± 24,09 ng E1S/mg Krea. Speichel und Milchkonzentrationen unterscheiden sich nicht signifikant zwischen den beiden Gruppen. Die gemessenen Konzentrationen von P4, E1S und RLN im Blut bzw. PdG und E1S im Urin stimmen mit den Ergebnissen früherer Untersuchungen überein und können somit als Trächtigkeitsmarker bestätigt werden. Dies ist die erste Arbeit, die trächtigkeitsassoziierte Hormone in Speichel und Milch von Alpakas untersucht. Während die P4 Bestimmung in Milch sowie die Bestimmung von PdG und E1S in Urin geeignete Alternativen darstellen, ist Speichel für eine Trächtig-keitsdiagnostik beim Alpaka ungeeignet. Die Nutzung von Milch und Urin zur Trächtigkeitsdiagnose stellt insofern eine Vereinfa-chung der derzeitig gängigen Methoden (u. a. Blutprogesteron) dar, als dass der Besit-zer das Probenmaterial selbst gewinnen kann und dies mit erheblich weniger Stress für die Stuten verbunden ist. Die Bestimmung von P4 in Milch und PdG in Urin stellen so-mit geeignete Alternativen zur Frühdiagnostik im ersten Trächtigkeitsmonat dar, da zu diesem Zeitpunkt eine transabdominale Ultraschalluntersuchung noch nicht aussage-kräftig ist. Die vorliegende Arbeit leistet einen Beitrag, um die noch vergleichsweise kleine vor-handene Datenbank zur Endokrinologie der Reproduktion bei NWK zu erweitern. / Aims of the present study were the measurement of pregnancy-associated hormones progesterone (P4), pregnanediol-glucuronide (PdG), relaxin (RLN) and oestrone sul-phate (E1S) in saliva, milk and urine of pregnant and non-pregnant alpacas, to compare to their respective blood concentrations and to assess their potential use for pregnancy diagnosis. Samples were obtained over a course of two years from 36 female alpacas of 6 private alpaca breeders in Saxony (Germany) before mating and at different stages throughout pregnancy (confirmed by ultrasonography). Hormone concentrations in serum, plasma, saliva, urine and milk samples were determined using enzyme immunoassays (EIA). Some milk samples were also tested using a commercial on-farm P4 kit which is de-signed for dairy cattle. Concentrations of P4 increased significantly from basal values in non-pregnant alpacas of 0.35 ± 0.04 ng/ml to 2.94 ± 0.11 ng/ml in plasma (and from 0.26 ± 0.03 to 2.87 ± 0.10 ng/ml in serum) in pregnant animals. Milk and urine concentrations of P4 were sig-nificantly higher in pregnant alpacas: Values increased from basal 0.83 ± 0.06 ng/ml to 4.09 ± 0.38 ng/ml in milk and from 0.29 ± 0.04 ng P4/mg Cr to 0.60 ± 0.06 ng P4/mg Cr in urine. While PdG concentrations in urine were significantly higher in pregnant (152.73 ± 17.37 ng PdG/mg Cr) than in non-pregnant animals (26.70 ± 2.80 ng PdG/mg Cr), there were no differences in concentrations of P4 or PdG in saliva. The on-farm milk P4 test kit showed a sensitivity of 90% for diagnosis of pregnancy and a specificity of 69% for non-pregnancy. RLN concentrations in blood increased significantly after the 2nd month from basal 1.65 ± 0.56 ng/ml to 11.69 ± 2.31 ng/ml in plasma and from 0.95 ± 0.30 ng/ml to 16.23 ± 3.05 ng/ml in serum, whereas there were no differences in milk, saliva and urine between pregnant and non-pregnant animals. Hormone concentrations of E1S increase during the last month of pregnancy: Blood concentrations rise from basal values of 0.59 ± 0.07 ng/ml to 3.43 ± 0.55 ng/ml in plasma and from 0.32 ± 0.02 ng/ml to 2.16 ± 0.43 ng/ml in serum; urine concentrations from 6.14 ± 0.53 ng E1S/mg Cr to 104.03 ± 24.09 ng E1S/mg Cr. There were no sig-nificant differences in E1S concentrations in saliva and milk between pregnant and non-pregnant alpacas. Values of P4, E1S and RLN in blood as well as PdG and E1S in urine are comparable to previous reports in alpacas and therefore can be confirmed as an indicator for preg-nancy. This is the first study to include determination of pregnancy associated hormones in saliva and milk of alpacas. However, saliva seems to be unsuitable for pregnancy di-agnosis in alpacas, whereas P4 in milk, as well as PdG and E1S in urine seem to be adequate tools. The use of milk and urine would simplify pregnancy diagnosis in alpacas since, in con-trast to the current methods (e.g. blood P4 concentration and ultrasonography), the owners themselves can take the samples. The avoidance of blood sampling results in a considerable stress reduction for the animals and therefore reduces the risk for potential loss of pregnancies. The measurements of P4 in milk and PdG in urine are useful alternatives to pregnancy diagnosis, especially during the first month of pregnancy, when transcutaneous ultrasonography is not yet reliable. This work adds information to the comparatively small database for camelid reproduc-tive endocrinology.

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.

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

Speichelglykane können einerseits spezifisch an bakterielle Lektine binden und damit deren Adhäsion an orale Oberflächen vermitteln, andererseits eine Antiadhäsion bedingen. Sie stellen ein Schutzsystem für orale Oberflächen dar. Bei vorhandener Karies bzw. Parodontitis ist die Konzentration bestimmter Glykokonjugate verändert. Ziel dieser Studie war es, die Reaktivität der Glandulae majores bzgl. ihrer Sekretion von Glykanen in Abhängigkeit einer experimentellen Gingivitis zu ermitteln. 14 gesunde Probanden enthielten sich 9 Tage der Mundhygiene. Neben der Erhebung des PBI und QH wurde drüsenspezifisch Speichel gewonnen. Die Konzentrationen an die Lektine PNA, GS1, VVA, SNA und AAA bindender Komponenten und deren drüsenspezifische Sekretionsraten wurden bestimmt. Bei allen Probanden stiegen PBI und QH im Versuchsverlauf signifikant an. Gleiches galt für die Speichelmenge nach Stimulation sowie zum Ende der Kontrollreihe. Die Konzentrationen der verschiedenen Glykane verhielten sich unabhängig von der Speichelmenge und unabhängig voneinander. Meist ergab sich eine erhöhte Glykansekretion spezifisch für das untersuchte Lektin. Neben dem Konzentrationsgefälle der einzelnen Drüsen war auch eine Verschiebung nach erfolgter Stimulation zu beobachten. Da genetische und externe Einflüsse für diese Studie weitgehend ausgeschlossen werden konnten bzw. als konstant einzuordnen waren, darf die Veränderung als Reaktion auf die orale Bakterienbelastung angesehen werden. Der Rückgang bestimmter terminaler Strukturen könnte als Folge der vermehrten Synthese anderer, in Bezug auf die veränderte Bakterienflora effektiverer Speichelbestandteile eingeordnet werden. Basierend auf dem Modell, dass freie Glykane die Adhäsion von Mikroorganismen inhibieren können, ließe sich die gemessene Reaktion der Speicheldrüsensekretion als ein gesteigerter Schutzmechanismus im Sinne einer ´first line of defence´ interpretieren. Dieser könnte z.B. in Bezug auf Prophylaxe und Therapie genutzt werden. / Salivary glycans can bind specificly to bacterial lectins. Consequently, bacterial adhesion to oral surfaces is mediated or inhibited by glycans. It is known that the concentration of certain glycans changes in the presence of caries or periodontitis. Therefore this study examines the reactivity of the major salivary glands with respect to the secretion of glycans as conditioned by an experimentally induced gingivitis. 14 healthy subjects refrained from all oral hygiene measures for 9 days. On 5 days a plaque and bleeding index as well as pure glandula saliva with and without stimulation were obtained. The collected salivary samples were examined for their concentration of certain structures that bind to the lectins ´PNA´, ´GS1´, ´VVA´, ´SNA´ and ´AAA´. All subjects developed a gingivitis as measured by the plaque and bleeding index. Salivary flow increased after stimulation and compared to baseline at the end of the trial. The concentration of glycans was neither related to one of the glands nor to the salivary flow. Besides to the differentials of concentration after stimulation there was no symmetrical development between the concentrations of salivary lectin-specific components compared one lectin to another. Genetic and external influences could be largely excluded or considered to be stable during the trial. Therefore the observed results can be regarded as a reaction to the increased bacterial load. The decrease of certain terminal structures in saliva might be explained by a raised synthesis of other components, which are more effective in defending the body against bacterial adhesion. The observed changes in salivary secretion might be interpreted as a mechanism in order to protect the human organism within the meaning of a ´first line of defence´. This mechanism would be able to respond more quickly than the immune system and might be used in future, for example, for preventive and therapeutical strategies.

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.

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.