La fonction de reproduction est sous le contrôle de la neurohormone hypothalamique GnRH qui régule la synthèse et la libération des gonadotropines hypophysaires. La GnRH agit par l’intermédiaire d’un récepteur couplé aux protéines G exprimé à la surface des cellules gonadotropes, le récepteur de la GnRH (RGnRH). Ce récepteur, chez les mammifères, a la particularité d’être dépourvu de queue C terminale ce qui le rend insensible aux systèmes classiques de désensibilisation. Ainsi, les mécanismes qui régulent l’efficacité et la spécificité de sa signalisation demeurent mal connus. Nous avons recherché des partenaires d’interaction du RGnRH, jusqu’alors inconnus, avec l’idée que ces protéines en interagissant avec les domaines intracellulaires du récepteur influenceraient son couplage aux voies de signalisation. Nos travaux ont permis d’identifier le premier partenaire d’interaction du RGnRH : la protéine SET. Par des expériences de « GST pull down », nous avons montré que SET interagit directement avec le RGnRH via le premier domaine intracellulaire du récepteur. Cette interaction implique des séquences riches en acides aminés basiques sur le récepteur et les domaines N- et C-terminaux de SET. Nous avons également montré, par co-immunoprécipitation, que le RGnRH dans sa conformation native interagit avec la protéine SET dans les cellules gonadotropes alphaT3-1 et, par immunocytochimie, que les deux protéines colocalisent à la membrane plasmique. En développant au laboratoire des outils biosenseurs permettant de mesurer avec une grande sensibilité et en temps réel les variations intracellulaires de calcium et d’AMPc, nous avons mis en évidence que le RGnRH se couple non seulement à la voie calcique mais aussi à la voie AMPc dans la lignée alphaT3-1, apportant pour l’AMPc la première démonstration d’un tel couplage. En utilisant différentes stratégies expérimentales visant à diminuer ou au contraire favoriser l’interaction du récepteur avec SET (ARN antisens, peptide correspondant à la première boucle intracellulaire du récepteur, surexpression de SET), nous avons montré que SET induit une réorientation de la signalisation du RGnRH de la voie calcique vers la voie AMPc. Nos résultats concernant l’activité du promoteur du gène du Rgnrh nous conduisent à postuler que SET pourrait favoriser l’induction par la GnRH de gènes régulés via la voie AMPc et notamment celui codant le RGnRH. Nos travaux mettent également en évidence que la GnRH régule non seulement l’expression de la protéine SET dans les cellules gonadotropes mais aussi son degré de phosphorylation favorisant ainsi sa relocalisation dans le cytoplasme des cellules alphaT3-1. Ceci suggère que la GnRH exerce une boucle de régulation permettant d’amplifier l’action de SET sur la signalisation de son propre récepteur. Enfin, nous avons mis en évidence que l’expression de SET est fortement augmentée dans l’hypophyse au moment du prœstrus chez le rat, apportant ainsi la première démonstration d’une variation de SET dans un contexte physiologique. Étant donné que le couplage du RGnRH à la voie de signalisation AMPc est augmenté au moment du prœstrus, nos résultats suggèrent que SET pourrait jouer un rôle important in vivo en favorisant ce couplage à ce stade particulier du cycle œstrien. / Reproductive function is under the control of the hypothalamic neurohormone GnRH, which regulates the synthesis and the release of pituitary gonadotropins. GnRH acts on a G-protein coupled receptor expressed at the surface of pituitary gonadotrope cells, the GnRH receptor (GnRHR). This receptor, in mammals, is unique because it is devoided of the C terminal tail, which makes it insensitive to classical desensitization processes. Therefore, the mechanisms that regulate the efficacy and the specificity of its signaling are still poorly known. We searched for interacting partners of GnRHR with the idea that these proteins by interacting with the intracellular domains of the receptor could influence receptor coupling to its signaling pathways. Our work identified the first interacting partner of GnRHR: the protein SET. By GST pull down assays, we showed that SET interacts directly with GnRHR through the first intracellular loop of the receptor. This interaction involves sequences enriched in basic amino acids in the receptor and both N- and C terminal domains of SET. We also showed, by co-immunoprecipitation, that GnRHR in its native conformation interacts with the endogenous SET protein in gonadotrope alphaT3-1 cells and, by immunocytochemistry that the two proteins colocalize at the plasma membrane. By developing in the laboratory biosensors tools that allow to measure with high sensitivity and in real-time intracellular variations in calcium and cAMP concentrations, we demonstrated that GnRHR couples not only to the calcium pathway but also to the cAMP pathway in alphaT3-1 cell line, providing for cAMP the first demonstration of such coupling. Using several experimental strategies to reduce or increase receptor interaction with SET (small interfering RNA, peptide corresponding to the first intracellular loop of the receptor, overexpression of SET), we have shown that SET induces a switch of GnRHR signaling from calcium to cAMP pathway. Our results concerning the activity of the Gnrhr gene promoter led us to postulate that SET could favor the induction by GnRH of genes regulated through the cAMP pathway, notably those encoding the GnRHR. Our study also showed that GnRH regulates not only SET protein expression in gonadotropes, but also its phosphorylation level leading to its relocation in the cytoplasm of alphaT3-1 cells. This suggests that GnRH induces a regulatory loop to amplify SET action on signaling of its own receptor. Finally, we demonstrated that SET expression is markedly increased in the pituitary gland at prœstrus in female rats, providing the first demonstration of a variation of SET expression in a physiological context. Given that GnRHR coupling to the cAMP pathway is increased at prœstrus, our results suggest that SET may play an important role in vivo by promoting such coupling at this particular stage of the estrus cycle.

No description available.

No description available.

Die vorliegende Arbeit beschäftigt sich mit der molekularen Struktur der amyloiden Aggregate von Antagonisten des Gonadotropin-Releasing Hormones (GnRH) und Analoga des Growth Hormone-Releasing Hormones (GHRH) sowie deren schrittweiser Bildung und der Inhibierung dieses Prozesses durch verschiedene Polyphenole. Die dabei erhaltenen Erkenntnisse werden genutzt, um pharmazeutisch relevante Depotformulierungen der genannten Peptide zu generieren und deren Freisetzungsverhalten zu untersuchen. Die 3D Strukturen der Peptidmonomere werden basierend auf zweidimensionalen NMR-Experimenten aufgeklärt. Zur Untersuchung der einzelnen Teilschritte der Aggregation dienen zahlreiche strukturanalytische Methoden, u.a. zeitaufgelöste Fluoreszenzspektroskopie (TCSPC), Untersuchungen mit strukturselektiven Sonden sowie TEM-Messungen. Ein Schwerpunkt liegt auf der Sekundärstrukturanalyse durch Bandenformanalyse von ATR-FTIR-Banden, um die Ausbildung der amyloid-typischen β Faltblattstrukturen detailliert zu beschreiben. Aus den Ergebnissen wird für GnRH-Antagonisten ein fibrilläres Aggregatmodell mit paralleler β Faltblattstruktur abgeleitet, dass zwei voneinander unterscheidbare β Faltblattbereiche enthält. Mithilfe einer eigens entwickelten Kongorot-Titrationsmethode wird der amyloid-aggregierte Anteil der Peptide in Gegenwart verschiedener Partnermoleküle untersucht. Dabei zeigt sich u.a., dass Poly-L-Glutamat nur eine der beiden β Faltblattstrukturen schwächen kann, wohingegen eine Gallotanninmischung aus Rhus chinensis die Amyloidbildung vollständig unterdrückt. Durch umfangreiche Arbeiten zur Trennung und Charakterisierung der polyphenolischen Komponenten der Gallotanninmischung werden Untersuchungen zu Struktur-Eigenschaftsbeziehungen ermöglicht. Unter Nutzung dieser Erkenntnisse werden pharmazeutisch relevante Formulierungen aus den Peptiden und Partnermolekülen hergestellt und mit einer speziell entwickelten Dialyse-Liberationsmethode auf ihr Freisetzungsverhalten untersucht. Da der aggregationsinhibierende Effekte der Polyphenole diffusionsbedingt schnell verloren geht, wird schließlich die Synthese von mit Polyphenolen modifizierten Chitosanen und deren erfolgreicher Einsatz zur Komplexierung der Peptide beschrieben, wodurch erstmals eine Formulierung zur Verfügung steht, welche die Aggregation des Peptidwirkstoffes verhindert und gleichsam eine kontrolliert verzögerte Freisetzung ermöglicht. / The present study addresses the molecular structure of the amyloid type aggregates of GnRH antagonists and GHRH analogs, their stepwise formation and the inhibition of this process by various polyphenols. The findings thus obtained are used to prepare pharmaceutically relevant drug delivery systems of the peptides and to study the corresponding release behavior. The 3D structure of the peptide monomers are elucidated by two-dimensional nmr experiments. Numerous structure analytical approaches are applied to obtain further insight into the partial steps of the aggregation process, e.g. TCSPC, experiments with structure selective probes and TEM. Emphasis is placed on the investigation of the secondary structure by band shape analysis of amide bands obtained by ATR-FTIR spectroscopy in order to analyze the formation of amyloid type β sheet structures. Based on the analytical results, a fibrilar aggregate model is described, which contains two distinguishable β sheet structures. Using a newly developed congored titration method, the amyloid content of the peptides is examined in the presence of various additives. Whereas poly-L-glutamate is only capable of weakening one of the two β sheet structures, a crude tannic acid mixture from Rhus chinensis may suppress the aggregate formation completely. Extensive efforts are made to enable experiments targeting the structure-activity relationships of the polyphenolic components of the tannic acid mixture. With this in mind, pharmaceutically relevant formulations of the peptides and several additives are prepared and their release properties investigated using a newly developed dialysis liberation device. Since the aggregation inhibiting effect is lost quite fast due to the diffusive loss of the polyphenolic compounds, furthermore the synthesis of chitosan, which is covalently modified with the polyphenlic components, and its successful application for the generation of drug delivery systems of the peptides is described.

Le Syndrome de Down, aneuploïdie la plus courante, a pour cause première la présence d'un chromosome 21 surnuméraire. L'établissement de cartes de corrélation génotypes/phénotypes chez les patients atteints du Syndrome de Down a permis de mettre en évidence la kinase DYRK1A, codée par le gène DYRK1A localisé dans la région DCR-1 du chromosome 21, comme candidat pouvant être impliqué dans l'apparition d'un retard mental. Comprendre le rôle et la régulation de DYRK1A est donc essentiel et pour cela, utiliser un test fiable de mesure d'activité de l'enzyme est indispensable. Nous avons développé une nouvelle méthode de dosage de l'activité kinase de DYRK1A utilisant la chromatographie en phase liquide à haute performance (HPLC). Cette méthode s'est révélée très sensible et peu coûteuse. En utilisant cette nouvelle méthode, nous avons confirmé les principales données obtenues in vitro sur l'activité de DYRK1A. Nous avons également caractérisé le comportement d'inhibiteurs connus de DYRK1A (harmine) et confirmé les résultats rapportés dans la littérature. En collaboration avec l'équipe du Dr. Dodd nous avons criblé des dérivés hétérocycliques azotés de faible poids moléculaire, inhibiteurs potentiels de DYRK1A. Enfin, le test d'activité a été utilisé ex vivo sur des extraits de cerveau de souris. Nos résultats indiquent que cette nouvelle méthode de dosage d'activité kinase est spécifique, reproductible et rapide. Elle peut potentiellement s'appliquer à d'autres kinases, phosphatases et plus largement à d'autres enzymes catalysant une réaction de modification de protéines. La GnRH joue un rôle essentiel en régulant la sécrétion et la synthèse de LH et de FSH via des récepteurs spécifiques (GnRHR) exprimés à la surface des cellules gonadotropes hypophysaires. L'expression tissulaire spécifique du Gnrhr est contrôlée par une combinatoire bien définie de facteurs de transcription comportant trois acteurs majeurs, SF1, LHX3 et ISL1. Au contraire du Gnrhr, nous montrons que les séquences régulatrices d'Isl1 en amont du site d'initiation de transcription (TSS) sont insuffisantes pour diriger l'expression hypophysaire spécifique, suggérant l'existence de mécanismes additionnels. De fait, les régions régulatrices (ou promoteurs) ainsi que les "corps" des gènes sont altérés par des modifications épigénétiques. Les résultats, obtenus par immunoprécipitation de la chromatine, montrent que dans les lignées cellulaires exprimant Isl1, notamment les cellules gonadotropes, Isl1 est complexé avec des histones H3 triméthylées sur la Lys4 (H3K4Me3) au niveau du TSS, une marque d'histones corrélée avec les gènes actifs. En revanche, dans les lignées cellulaires où Isl1 est silencieux, il est complexé avec des histones H3 triméthylées sur la Lys27, marque liée à la répression des gènes. On observe cette même corrélation au niveau du TSS du Gnrhr. De plus, notre étude suggère que la méthylation de l'ADN, en amont de l'îlot CpG est inversement corrélée à l'activité de ce gène. Ces données suggèrent que les modifications épigénétiques sont essentiellement responsables de l'expression hypophysaire spécifique d'Isl1contrairement à l'expression du Gnrhr qui semble principalement dépendante de la présence de facteurs de transcription tissulaires spécifiques majeurs. Mots clefs : ISL1, récepteur du GnRH, antéhypophyse, régulation épigénétiques, modifications des histones, méthylation de l'ADN, protéine LIM à homéodomaine, cellules gonadotropes.

The control of reproductive cycles in higher primates is largely dependent on negative and positive feedback mechanisms by both steroidal and non-steroidal substances of the ovaries which regulate the function of hypothalamo-pituitary system. To gain insights into the role of INH A, the non steroidal ovarian hormone in the feedback control of pituitary FSH secretion, studies were conducted to examine the interrelationships of hormones throughout the menstrual cycle of the bonnet macaque. The findings of chapter II provide a detailed description of endocrine hormone profile during the menstrual cycle of the bonnet macaques with special attention to the feedback role of INH A on pituitary FSH secretion. To characterize the endocrine profile of different hormones, both ovarian (E2, P4, INH A) and pituitary (FSH, LH) hormones were measured daily for more than 40 days. To further examine the site of secretion of INH A and its relationship with pituitary FSH dynamics, surgical lutectomy and pharmacological induction of luteolysis employing the third generation GnRH R antagonist, Cetrorelix (CET) studies were carried out in the subsequent experiments. The results obtained from these studies suggest that INH A and P4 secreted from the CL during luteal phase regulate pituitary FSH secretion. The selective rise in FSH observed during the late menstrual cycle and during menstruation (referred to as luteo-follicular transition), as has been reported previously in higher primates, considered necessary for initiation of follicular growth and recruitment of follicles for ensuing menstrual cycle was characterized in the monkey. Surgical lutectomy and induction of luteolysis by CET experiments suggested that increased GnRH secretion is essential for this selective rise in FSH following withdrawal of inhibition by P4 and INH A. In clinical cases of reproductive ageing, the shortened follicular phase in human females has been identified to be the result of occurrence of early onset of FSH rise during the luteal-follicular transition period. The cause(s) of declining fertility with age in women who still have regular menstrual cycles are not clear, but issues of relationship between dysregulation of selective FSH rise in the late luteal phase and associated infertility could be examined using bonnet monkey as a model system. INH A is secreted in significant quantities by CL in higher primates and the feto placental unit suggesting its importance during fertility and pregnancy. Apart from the negative feedback regulation of pituitary FSH secretion, the complete repertoire of actions of this hormone during pregnancy is yet to be fully understood. The data presented in this thesis is the first comprehensive report showing the endocrine hormone profile of gonadotropins and ovarian hormones including INH A throughout the menstrual cycle of the bonnet macaque. The characterization of INH A profile in bonnet monkey will be of significant value for studies examining the role of INH A in higher primates. Dimeric inhibin has been suggested to be important for regulation of fertility and reproductive functions. Also, inhibin-α (one of the subunits of the dimeric protein) knock out mice model has provided convincing evidence that it acts as a tumour suppressor. A great deal of new information has been generated in recent years regarding the potential clinical usefulness of monitoring inhibin levels in blood and biological fluids in gynaecological diseases, pathological pregnancies and other disorders. Emerging clinical roles of inhibin have made INH A an important candidate molecule to study its molecular regulation. The results presented in chapter II suggested that LH regulates luteal INH A secretion (induction of luteolysis by CET administration experiment). As a first step towards understanding molecular regulation of inhibin-α expression in the macaque CL, in silico promoter analysis of macaque inhibin-α was performed and it revealed several transcriptional factor binding sites that were conserved across species. In rats FSH up regulates while preovulatory LH surge suppresses inhibin-α mRNA expression in the ovary and this suppression has been suggested to be necessary for occurrence of secondary FSH surge during metestrus. To address differential regulation of inhibin-α by LH and FSH in rat ovary during the periovulatory period, studies employing immature rats were carried out and the results are presented in chapter III. The results suggest that immature rat ovaries respond to exogenous gonadotropins in terms of LH signaling (cAMP production), luteinization (P4 production) and as well induction of ICER expression required for repression of inhibin-α subunit expression. PDE4 inhibitor (rolipram) treatment enhanced the ovarian cAMP concentrations suggesting that PDE4 play a major role in controlling intraovarian cAMP concentrations in rat ovaries. However increased cAMP concentrations did not appear to up regulate the ICER expression at the time point examined in this study. In higher primates time course of second FSH surge and continued synthesis and secretion of INH A in the CL are different from non primate species. In the monkey, the second FSH rise occurs during the late luteal phase and experiments have been carried out to examine the regulation of inhibin-α subunit expression by ICER. Expressions of ICER (mRNA/protein) and INH A were examined during different stages of CL and the results indicated no clear inverse relationship between the ICER and inhibin-α mRNAs. With no conclusive role for the ICER in regulating luteal inhibin-α observed in the study, the role of transcriptional activators in the regulation of inhibin-α like GATA4, SF-1, β-catenin were further examined. Since luteal INH A secretion was dependent on pituitary LH as determined earlier in chapter II, expressions of transcriptional activators were examined in CL of different stages and also during induced luteolysis and the results are described in chapter IV. In conclusion, our results indicate cross talk between WNT, cAMP and P38 MAP kinase signaling pathways in the regulation of luteal INH A secretion. The pituitary gonadotropin, LH, is the primary luteotropin in primate species acting to maintain the structure and function of the CL during the menstrual cycle. However whether the actions of LH are direct or mediated by local factors such as P4 remain unknown. Moreover, P4 secretion which is dominant during luteal phase has any role in regulating CL structure and function is not clearly defined. To address these and issues concerning P4 actions, initially, experiments were performed in the rat model to study the importance of P4 in the regulation of ovarian functions. An antiprogestin, RU486, was employed as a tool to uncover the PR regulated pathways during ovulation in rats and the findings are presented in the chapter V. The results indicated that blockade of PR action by RU486 during gonadotropin-induced superovulation resulted in inhibition of follicular rupture and ovulation in immature rats. Further to understand the downstream effectors of PR action, and to identify the candidate target genes of PR activation, semi-quantitative RT-PCR and western blot analyses were performed. The results obtained indicated that betacellulin, a member of EGF family and MMP-9 a proteolytic enzyme, were markedly repressed in response to RU486 treatment in rat ovaries. Also, the down stream pathway of EGF signaling leading to activation of ERK was markedly repressed in RU486 treated ovaries. It was next examined what role the P4/PR system has in the regulation of CL structure and function. Surprisingly, PR expression is absent in CL of rats, while it is present in higher primates. Experiments were carried out to examine intracrine actions of P4 in the regulation of CL structure and function in monkeys. The recently reported model system of induced luteolysis yet capable of responsive to trophic support from the laboratory provided an ideal opportunity to examine direct effects of P4 on structure and function of CL in the monkey. A series of pilot experiments were carried out in monkeys experiencing summer amenorrhea, to determine dose and mode of administration of exogeneous P4 to simulate mid luteal phase circulating P4 concentrations in monkeys subjected to induced luteolysis. Based on the results of pilot experiments, implantation of Alzet pumps containing 97.5mg of P4 was selected for maintaining mid luteal phase P4 concentrations. The microarray data of induced luteolysis previously deposited by the laboratory in NGBI’s gene expression omnibus were mined for identification and validation of differentially expressed genes of PR and its target genes following LH depletion and LH replacement experiments. Expressions of PR, PR cofactors and expressions of PR downstream target genes through out the luteal phase and in CL from day1 of menses were also examined. Analysis of expressions of genes revealed that of the 45 genes identified to be regulated by LH treatment, 4 genes were found to be responsive to P4, and 14 were identified to be responsive to both P4 and LH. Morphology of CL tissue sections revealed that P4 treatment appeared to have reversed the induced-luteolysis changes. In another experiment, implantation of P4 during late luteal phase (i.e., the period of declining P4 concentrations) for 24h caused changes in expressions of genes associated with tissue remodeling and morphology of luteal cells. Taken together, the results suggest that induced luteolysis plus P4 replacement model is suitable for assessing the effects of P4 on CL function. The results also suggest that CL could serve as target tissue for examining the genomic and non genomic actions of P4. In summary, studies carried out in the present thesis provides a comprehensive endocrine hormone profile throughout the menstrual cycle of the bonnet monkey with special emphasis on time course of INH A and FSH secretion which is very useful for future investigations. Studies have been carried out in rats and monkeys with different experimental model systems to address molecular mechanisms underlying inhibin-α regulation in the ovary in general and CL in particular. Experimental findings in monkeys could help elucidate the underlying molecular nature of CL functionality and extrapolate to understand luteal insufficiency and infertility producing conditions in humans. Also different model systems have been validated to examine the actions of P4/PR system in rats and monkeys and more importantly to address the direct effects of P4 upon monkey CL structure and function were established. Future investigations based on findings of these studies should help clarify relative roles for LH and P4 during maintenance of CL function and luteolysis.

Thesis by publication -- 8 co-authored articles. / Thesis (PhD)--Macquarie University, Division of Environmental and Life Sciences, Department of Biological Sciences. / Includes bibliographical references. / Preface -- Management issues of urban common brushtail possums (Trichosurus vulpecula): a loved or hated neighbour -- Effects of deslorelin implants on reproduction in the common brushtail possum (Trichosurus vulpecula) -- Brushtail possums (Trichosurus vulpecula) in metropolotan Sydney: population biology and response to contraceptive implants -- Strategic survey for Toxoplasma gondii and Neospora caninum in the common brushtail possum (Trichosurus vulpecula) from urban Sydney, Australia -- Leptospirosis serology in the common brushtail possum (Trichosurus vulpecula) from urban Sydney, Australia -- Conclusions. / The common brushtail possum (Trichosurus vulpecula) is indeed a common inhabitant of many Australian citites, and one of the few marsupials that has adapted well to the urban environment. Their close proximity to people provides a great opportunity to experience native wildlife in the backyard, however, their utilization of house roofs, bold behaviour and appetite for garden plants often leads to conflict with householders. Population numbers are sufficiently high to require ongoing management to minimise negative impacts for humans and brushtail possums alike in a socially acceptable manner. The aim of this thesis was to identify current management issues and address the need for improved and novel management strategies. The potential of slow-release implants, containing the GnRH agonist deslorelin, as a contraceptive agent for brushtail possums was tested on a captive population. Males appeared resistant to treatment, but deslorelin was found to inhibit reproduction in female brushtail possums for at least one breeding season, making it a promising tool to control fertility in some wild populations. A further aim was to trial deslorelin implants on a wild urban population, to collect more information about the urban biology of this species and to point out issues which have previously not been addressed. Close proximity and interaction of urban brushtail possums with humans and their domestic animals can increase the risk of disease exposure and transmission and influence the health of wild populations. Serosurveys showed that animals were readily exposed to Leptospira spp. and Toxoplasma gondii. This thesis also provides the first data on brushtail possum dispersal in urban areas, knowledge which is highly relevant to the development of management strategies such as fertility control. The findings from this research broaden our knowledge about urban brushtail possums and should assist wildlife authorities in developing alternative or improved management procedures. / Mode of access: World Wide Web. / xxv, 287 p. ill., maps

Die vorliegende Arbeit beschäftigt sich mit der Untersuchung und Beschreibung der Eigenschaften der synthetischen Dekapeptide Cetrorelix und Ozarelix durch analytische Methoden und computergestützte Modellierung. Diese Moleküle sind hydrophobe, aggregierende Antagonisten des Gonadotropin-Releasing-Hormons (GnRH). Zusätzlich wurden amyloidbildende Peptidstrukturen als Modelle für die Assoziationsprozesse in hydrophoben Peptiden untersucht und visualisiert. Die intrinsische Fluoreszenz der GnRH-Antagonisten und zusätzlich der Peptide Teverelix und D-Phe6-GnRH sowie von verkürzten Fragmenten des Cetrorelix wurde untersucht. Ein Strukturmodell für die Beschreibung der Aggregation der Dekapeptide wurde erarbeitet. Der Aufbau eines Rechenclusters durch das Einbinden der Computer am Lehrstuhl in ein Linux-System zur Verteilung von Rechenprozessen über das Netzwerk ermöglichte die Bereitstellung der notwendigen Leistung zur Realisierung der Berechnungen. Es wurden Werkzeuge zur Modellierung der solvatisierten Aggregate von Peptiden ohne eindeutige Vorzugsstruktur programmiert und in ein Docking-System für beliebige Moleküle eingebunden. Verwendet wurde das Kraftfeld MMFF94 mit einer Erweiterung durch ein Verfahren zur dynamischen Berechnung von Partialladungen in Molekülstrukturen. Solvatisierte Aggregate der Dekapeptide und von bekannten amyloidbildenden Strukturen wurden modelliert (Docking). Berechnet wurden als aggregierend beschriebene Sequenzen und entsprechende Vergleichsstrukturen des Calcitonins, des Insel-Amyloid-Polypeptides, des beta2-Mikroglobulins, des Amyloid-beta-Proteins, des Lactoferrins und weitere Modellpeptide. Die wesentlichen Wechselwirkungen während der Aggregation konnten schließlich anhand von Dynamik-Simulationen der faltblattartigen Dimere des Cetrorelix und Ozarelix beschrieben werden. So wurden die Prozesse der hydrophoben Assoziation und Stabilisierung durch Wasserstoffbrücken von Peptiden veranschaulicht und auf molekularer Ebene erfolgreich analysiert. Die Visualisierung der erhaltenen Modellierungsergebnisse erfolgt durch die Darstellung der Strukturen und Dynamik-Simulationen als interaktive 3D-Modelle in einem für diese Arbeit aufgebauten Internetauftritt. / This work discusses the analysis of the aggregation properties of the gonadotropin releasing hormone antagonists Cetrorelix, Teverelix, Ozarelix and of small amyloid forming model peptides by analytical fluorescence spectroscopy and molecular modelling. A high performance linux compute cluster was developed for calculation of molecular structures. Solvated aggregate clusters of peptides without defined secondary structure were modelled by molecular mechanics methods (forcefield mmff94) in combination with an advanced charge equilibration and docking technique. Molecular dynamics of solvated peptide dimers were implemented and the role of hydrophic association and hydrogen bond formation in hydrophobic peptide aggregates was explained. Finally, an aggregation model for the directed association of hydrophobic peptides is presented. The modelling results, 3d structures and dynamic simulations are visualized in an interactive web material.

Die vorliegende Arbeit beschäftigt sich mit der Untersuchung und Beschreibung der Eigenschaften der synthetischen Dekapeptide Cetrorelix und Ozarelix durch analytische Methoden und computergestützte Modellierung. Diese Moleküle sind hydrophobe, aggregierende Antagonisten des Gonadotropin-Releasing-Hormons (GnRH). Zusätzlich wurden amyloidbildende Peptidstrukturen als Modelle für die Assoziationsprozesse in hydrophoben Peptiden untersucht und visualisiert. Die intrinsische Fluoreszenz der GnRH-Antagonisten und zusätzlich der Peptide Teverelix und D-Phe6-GnRH sowie von verkürzten Fragmenten des Cetrorelix wurde untersucht. Ein Strukturmodell für die Beschreibung der Aggregation der Dekapeptide wurde erarbeitet. Der Aufbau eines Rechenclusters durch das Einbinden der Computer am Lehrstuhl in ein Linux-System zur Verteilung von Rechenprozessen über das Netzwerk ermöglichte die Bereitstellung der notwendigen Leistung zur Realisierung der Berechnungen. Es wurden Werkzeuge zur Modellierung der solvatisierten Aggregate von Peptiden ohne eindeutige Vorzugsstruktur programmiert und in ein Docking-System für beliebige Moleküle eingebunden. Verwendet wurde das Kraftfeld MMFF94 mit einer Erweiterung durch ein Verfahren zur dynamischen Berechnung von Partialladungen in Molekülstrukturen. Solvatisierte Aggregate der Dekapeptide und von bekannten amyloidbildenden Strukturen wurden modelliert (Docking). Berechnet wurden als aggregierend beschriebene Sequenzen und entsprechende Vergleichsstrukturen des Calcitonins, des Insel-Amyloid-Polypeptides, des beta2-Mikroglobulins, des Amyloid-beta-Proteins, des Lactoferrins und weitere Modellpeptide. Die wesentlichen Wechselwirkungen während der Aggregation konnten schließlich anhand von Dynamik-Simulationen der faltblattartigen Dimere des Cetrorelix und Ozarelix beschrieben werden. So wurden die Prozesse der hydrophoben Assoziation und Stabilisierung durch Wasserstoffbrücken von Peptiden veranschaulicht und auf molekularer Ebene erfolgreich analysiert. Die Visualisierung der erhaltenen Modellierungsergebnisse erfolgt durch die Darstellung der Strukturen und Dynamik-Simulationen als interaktive 3D-Modelle in einem für diese Arbeit aufgebauten Internetauftritt. / This work discusses the analysis of the aggregation properties of the gonadotropin releasing hormone antagonists Cetrorelix, Teverelix, Ozarelix and of small amyloid forming model peptides by analytical fluorescence spectroscopy and molecular modelling. A high performance linux compute cluster was developed for calculation of molecular structures. Solvated aggregate clusters of peptides without defined secondary structure were modelled by molecular mechanics methods (forcefield mmff94) in combination with an advanced charge equilibration and docking technique. Molecular dynamics of solvated peptide dimers were implemented and the role of hydrophic association and hydrogen bond formation in hydrophobic peptide aggregates was explained. Finally, an aggregation model for the directed association of hydrophobic peptides is presented. The modelling results, 3d structures and dynamic simulations are visualized in an interactive web material.