L’acquisition de la fertilité chez les mammifères est le résultat d’un long processus de développement et de maturation de l’axe gonadotrope. Cette fonction cruciale à la survie des espèces est orchestrée par une poignée de cellules localisées au niveau de l’aire préoptique hypothalamique chez le rongeur, sécrétant la gonadotropin-releasing hormon (GnRH). La GnRH stimule la sécrétion de LH et de FSH par l’adénohypophyse, qui stimulent à leur tour les gonades. Les cellules à GnRH naissent dans l’épithélium voméronasal pendant le développement embryonnaire et migrent le long des axones voméronasaux pour atteindre l’hypothalamus. A la naissance le système est entièrement en place, toutefois il subira une phase de maturation avant d’atteindre la puberté, signant le début de la fertilité. Chez l’homme, un défaut de sécrétion de GnRH peut conduire à un hypogonadisme hypogonadotrope idiopathique (IHH) caractérisé par une subfertilité et une puberté retardée voire absente, ou même à un syndrome de Kallmann. Dans une grande partie des cas ce défaut de sécrétion est lié à un défaut de développement prénatal et à une diminution du nombre de neurones à GnRH dans dans l’hypothalamus. Depuis peu, la grande famille des semaphorines, déjà connues pour leurs effets chimiotactiques dans certains types cellulaires, et en particulier la semaphorine3A (Sema3A) via son récepteur la Neuropilin-1 (Nrp1), a été décrite comme un facteur indispensable au développement du système à GnRH et décrit comme un « gène Kallmann ». Toutefois son rôle spécifique dans les cellules à GnRH reste à élucider. Le premier objectif de ma thèse a donc été de déterminer le rôle de l’expression du récepteur Nrp1 dans les neurones à GnRH. Le suivi de la maturation sexuelle des animaux Gnrh::cre, Nrp1loxp/loxp (qui n’expriment pas la Nrp1 exclusivement dans les neurones à GnRH) a révélé l’apparition d’une puberté précoce et d’un phénotype de surpoids en comparaison aux animaux contrôles, corrélé à une accumulation des cellules à GnRH dans l’aire préoptique. L’étude de l’embryogenèse du système à GnRH chez ces animaux a démontré une augmentation du nombre de cellules à GnRH pendant leur migration. Nos résultats obtenus in vivo et in vitro suggèrent que la signalisation Nrp1 a un impact sur la survie des neurones à GnRH, et qu’elle module la motilité des cellules en migration et influe leur positionnement dans le cerveau. Le deuxième objectif de ma thèse a été d’étudier le rôle de l’exocytose dépendante du calcium et donc de la neurosécrétion dans les neurones à GnRH sur leur développement. Le suivi de la physiologie d’animaux Gnrh::cre; iBot, dont l’exocytose dépendante du calcium est abolit par clivage de protéine VAMP2/synaptobrevin 2 dans le neurone à GnRH, a révélé l’apparition de deux phénotypes distincts selon la pénétrance du transgène : un groupe ayant une puberté normale et un poids comparable aux animaux contrôles, et un groupe ayant une puberté retardée voire inexistante associé à un surpoids. Ces derniers présentent un IHH, une augmentation du tissu adipeux périgonadique et une hyperleptinémie, alors que la distribution des neurones à GnRH dans le cerveau n’est pas altérée. Ces données mettent en évidence le fait que l’activité de neurosécrétion dans les neurones à GnRH ne serait pas nécessaire pour leur développement embryonnaire, mais qu’elle pourrait jouer un rôle dans le maintien de l’homéostasie énergétique.Ces deux études mettent en avant un lien étroit entre axe gonadotrope et métabolisme énergétique chez les mammifères et ont dévoilés de nouveaux mécanismes qui pourraient être impliqués dans la physiopathologie de la reproduction chez l’homme. / Fertility in mammals is the result of a long development and maturation process of the hypothalamic-pituitary-gonadal axis. The reproductive function is orchestrated by a small population of neurons, located in preoptic area of hypothalamus in rodents, and releasing in a pulsatile manner Gonadotropin-releasing hormon (GnRH) in the portal blood vessels, where it is transported to the anterior pituitary gland. GnRH neuropeptide triggers synthesis and release of the gonadotropins LH and FSH, which in turn stimulates development and function of the gonads. GnRH neurons differenciate extracerebraly in the nasal placode and migrate from the vomeronasal organ to the forebrain along olfactory/vomeronasal nerves. At birth, the system is ready, however it will undergo a maturation phase before reaching puberty, signing the beginning of fertility. Deficiency in GnRH release can lead to idiopathic hypogonadotropic hypogonadism (IHH), characterized by a defect in sexual maturation and delayed or no puberty, or even to Kallmann syndrome when the IHH is associated with a deficit in the sens of smell. These phenotypes could be linked to a defect during GnRH neuron migration period and a decrease of GnRH cells located in hypothalamus after birth. Numerous studies have described the influence of different molecules on the migration of GnRH neurons. Recently, the semaphorin family, well known for its chemotactic effects in some cell types, and particularly the semaphorin3A (Sema3A), has been described by our laboratory as an essential factor for the guidance of GnRH neurons during embryogenesis, and characterized as a « Kallmann gene ». However, the role of Sema3A, and its specific receptor Neuropilin-1 (Nrp1) in GnRH neurons remains to be elucidated. The first objective of my thesis was to determine the role of the expression of Nrp1 in GnRH neurons. The analysis of sexual maturation in mice in which Nrp1 expression was selectively knocked out in GnRH neurons revealed a precocious onset of puberty and overweight compared to control littermates, correlated with an accumulation of GnRH neurons in preoptic area. The study of the development of the GnRH system during embryogenesis has shown an increased number of cells during migration. In vivo and in vitro data suggested the involvement of Nrp1 signaling pathway in the survival of GnRH neurons, the control of their motility during migration, and their final positioning in the brain.The second objective of my thesis was to study the role of Calcium-dependent exocytosis, and thus neurosecretion, in GnRH neurons on their development. The monitoring of Gnrh::cre; iBot animals, in which calcium-dependent exocytosis is abolished by cleavage of VAMP2/synaptobrevin2 protein in GnRH neurons, showed the distinction of two different phenotypes. A subpopulation of mice underwent normal puberty onset, with a similar bodyweight than control littermates, and the other one never reached puberty and developed overweight. The later animals exihibited IHH, increase of the volume of perigonadic fat tissue, and hyperleptinemia, with no alteration of GnRH neuron number and distribution. This data established that neurosecretion in GnRH neurons is not a prerequisite for their migration during embryonic development but revealed that it could play an important role in metabolic homeostasis.Together these two studies highlight an intriguing direct connection between GnRH neurons and energy metabolism in mammals as well as new mechanisms that could be implicated in reproductive physiopathology in human.

Electroconvulsive therapy (ECT) is one of the oldest and most effective treatments for depression; however, its biological underpinnings are poorly understood. Brain-derived neurotrophic factor (BDNF) and the hypothalamic-pituitary-adrenal (HPA) axis are two chemical messenger systems implicated in the antidepressant action and cognitive side effects of ECT. The Wistar-Kyoto (WKY) strain is a genetic model of depression that shows biological, cognitive, behavioural, and treatment-response abnormalities, making it potentially a useful model in which to investigate the underpinnings of the action of electroconvulsive stimulus (ECS: the amimal model of ECT). In addition, the WKY presents a potentially useful model for translational research on depression. The WKY strain is particularly valuable for the measurement of serum BDNF protein, for which the association with antidepressant treatments is much less clear (mostly stemming from investigations in humans) than that between brain BDNF and antidepressant treatments in rodent studies. The three studies presented add insight into the biological and behavioural effects of ECS. The first study (chapter 2) found no evidence of increased (R)-[11C]rolipram binding (an indirect marker of cyclic-adenosine monophosphate, cAMP) in the brain, despite significant increases of brain BDNF protein expression after repeated ECS. The second study (chapter 3) demonstrated the validity of the WKY strain in the investigation of ECS. Relative to Wistar controls, WKY showed similar antidepressant and cognitive effects (despite some abnormal behavioural responses), immediate but not sustained increases in brain BDNF protein, and a novel finding of increased extra-hypothalamic CRF after 5 daily ECS. The final study (chapter 4) demonstrated baseline strain differences in serum (WKY < Wistar) but not brain BDNF and, in both strains, no change in serum BDNF despite significant changes in brain BDNF after repeated ECS treatment. Preliminary results from a human pilot study investigating similar measures in a small group of people receiving ECT for depression are also presented. The results of this body of work advance our understanding of the activation and role of brain and serum measures of BDNF and the HPA axis in ECS/ECT, and raise important issues in the translation of research from basic science to the human condition of depression.

Depression occurs in about one third of patients with stroke and cardiac arrest. Hyperactivity of the stress system is the most commonly observed neuroendocrine change in major depressive disorder (MDD), which involves elevated levels in the cerebrospinal fluid of corticotropin-releasing hormone (CRH), a key stress neurohormone. Substantial evidence suggests that normalization of the stress system may be a requirement for successful treatment of MDD through region-specific changes in the mesocorticolimbic circuitry. Thus, alteration in the stress system may underlie the emotional and functional impairments observed following brain ischemic events. In addition, recent findings suggest that ischemic brain injury triggers a restorative process, creating a cerebral environment similar to that of early brain development, a period characterized by rapid neuronal growth and neuroplasticity, critical to optimize functional recovery of individuals post stroke. In particular brain-derived neurotrophic factor (BDNF), has been shown to play an important role in the pathophysiology of major depression and cerebral ischemia. However, whether CRH can mediate the expression of BDNF in the reparative process triggered by ischemic injury remains to be characterized. Therefore, the purpose of the current thesis is to characterize the effect of pharmacological blockade of CRH signaling at the onset of a global ischemic stroke, on emotional and cognitive behaviors, alteration in the neuroendocrine stress system, and markers of neuroplasticity including BDNF. To do this, an animal model of global cerebral ischemia with subsequent behavioral testing and postmortem brain analysis was used to determine underlying biochemical and behavioral changes modulated by CRH signaling following brain ischemia. This doctoral work will help elucidate the relationship between CRH and BDNF in the context of cerebral ischemia, and may provide insights for therapies targeting the stress system. These studies address considerations such as: the interplay between stress, neuroplasticity and emotionality, and whether global ischemia can affect mood via changes in the HPA axis response.

<p>Some individuals with alcohol use disorder (AUD) continue to drink because they have developed a habit in which they are not considering the consequences of their actions. Habitual actions persist despite changes in reward and are often studied using devaluation procedures. Stress hormones, such as corticotropin releasing factor (CRF), have been linked to AUD when examining binge-like drinking and withdrawal in rodents. Stress has been examined in the switch from goal-directed to habitual behavior, and CRF has often mimicked the effects of stress exposure. This study looked at the possible direct effects of CRF on habit expression in rats using an operant paradigm. Finding possible novel mechanisms of habit could create an avenue for future novel treatment options. Female and male Long Evans rats were trained on a variable interval schedule using sucrose as a reward. Rats then underwent devaluation procedures including both sensory-specific satiety and conditioned taste aversion (CTA) to test for habitual behaviors. Prior to an extinction session post-CTA, animals were treated with either 20 mg/kg R121919, a CRF1 receptor antagonist, or vehicle. A second extinction session was conducted where animals received the alternative treatment. Lever presses were recorded as a measure of goal-directed or habitual behavior. Sensory-specific satiety devaluation tests revealed that animals were not sensitive to devaluation. This was further supported by both post-CTA extinction sessions. R121919 had no effect on lever pressing in either devalued or valued groups. Further research is needed to explore how a CRF receptor antagonist may affect habit formation or the transition from goal-directed to habit behaviors. Future studies should also examine any possible interaction effects CRF may have with alcohol or stress on habitual behaviors.</p>

Gonadotropin-releasing hormone (GnRH) and its superactive analogues are currently being used in the treatment of a number of endocrine disorders, such as endometriosis, precocious puberty, infertility and prostatic cancer. Selection of these analogues for clinical use have been previously based on their activities in animal models. This thesis has therefore investigated the binding characteristics of the human GnRH receptor, in comparison to those of the rat receptor, as well as the activities of a number of GnRH analogues for stimulating luteinising hormone (LH) and follicle stimulating hormone (FSH) secretion from cultured human pituitary cells. The establishment of a human pituitary bioassay system has further made possible the investigation of the direct regulatory roles of GnRH and other neuropeptides in man. To date, such studies in man have been performed in vivo and are thus complicated by the simultaneous interactions of numerous modulators.

The control of prolactin secretion is a complex interaction of peptides and neurotransmitters acting either in an inhibitory or stimulating way to effect final secretion of this hormone from the lactotrope cell in the anterior hypothalamus. These factors may act either directly on the lactotrope cell or indirectly by changing either dopamine restraint of prolactin secretion or by modulating peptide substances or neurotransmitters higher up in the hypothalamus. Gonadal steroids may also modulate the effect of peptides or dopamine at the level of the lactotrope. Prolactin's major role in the female rat is one of milk production post - partum, nurturing the young. It probably also has other physiological functions and may play a part in the menstrual cycle although this is controversial. Certainly, pulsatile secretion of prolactin during the menstrual cycle is well established and in the luteal phase this is concomitant with the secretion of luteinizing hormone. Theories explaining the synchronous surges seen during this phase of the menstrual cycle have been proposed and GnRH has been implicated in the genesis of the concordance of these secretory spikes. Using a potent GnRH antagonist an experiment was undertaken to establish the role of GnRH by blocking this hypothalamic peptide and observing the effect that this had on luteinizing hormone, prolactin and follicle stimulating hormone. In the first part of the thesis the control of prolactin secretion is reviewed. In the following section, an experiment was performed using a potent GnRH antagonist. A dose response curve was established for the antagonist action on LH. Then a twice maximum dose of this peptide was administered to three subjects in the midluteal phase of the menstrual cycle and the response of LH, prolactin and FSH was measured. The results indicate that although the GnRH antagonist significantly blocked LH secretory peaks, this action was not observed for either prolactin or FSH. This result is perhaps at variance with previous data which suggested that GnRH was responsible for concordant secretory spikes of LH and prolactin in the midluteal phase of the menstrual cycle.

In der vorliegenden Arbeit sollte die Wirksamkeit von Altrenogest (Regumate®) und Gonadotropinen bzw. des GnRH-Analogons D-Phe6-GnRH im Rahmen eines Verfahrens zur Brunst- und Ovulationssynchronisation bei besamten, als ingravid detektierten Jung- und Altsauen überprüft werden. Die Untersuchungen wurden an insgesamt 265 Jung- und 542 Altsauen vorgenommen. Die Sauen wurden im Rahmen der ultrasonografischen Trächtigkeitskontrolle, die zwischen den Tagen 21 und 35 nach der ersten künstlichen Besamung durchgeführt wurde, als ingravid detektiert und einer von drei verschiedenen Versuchsgruppen (VG) zugeteilt: VG 1 (n = 490): Applikation von 4 ml Regumate®/Tier/Tag oral über 15 Tage; einmalige subkutane (s.c.) Injektion von 1.000 IE PMSG/eCG 24 Stunden nach letztmaliger Regumate®-Gabe; i.m. Applikation von 500 IE hCG 78 bis 80 h nach PMSG/eCG zur Induktion der Ovulation; VG 2 (n = 135): identisch zu VG 1, aber Gabe von 5 ml Regumate® und 800 IE PMSG; VG 3 (n = 182): identisch zu VG 2, aber Injektion von 50 µg D-Phe6-GnRH zur Induktion der Ovulation. Die Sauen wurden jeweils zweimal im Abstand von 24 und 40 Stunden nach hCG bzw. D-Phe6-GnRH künstlich besamt. Der Erfolg der Behandlungen wurde anhand der sonografischen Untersuchung der Ovarien kontrolliert. Untersuchungen erfolgten zum Zeitpunkt der Trächtigkeitsuntersuchung, am Ende der Regumate®-Behandlung, unmittelbar vor der ersten und unmittelbar nach der zweiten künstlichen Besamung. Trächtigkeits- (TR) und Abferkelrate (AFR) sowie Anzahl insgesamt und lebend geborener Ferkel wurden dokumentiert. Als jeweilige Kontrolltiere dienten Jung- bzw. Altsauen, die brunst- und ovulationssynchronisiert wurden, zur Erstbesamung anstanden und zeitgleich wie die Tiere der Versuchsgruppe besamt wurden. Die Auffindungsrate der Ovarien betrug 88,9 % zum Zeitpunkt der Trächtigkeitsuntersu-chung (TU) und 98,3 % am Ende der Regumate®-Behandlung. Während der Besamungen konnten bei allen untersuchten Tieren die Ovarien dargestellt werden. Zum Zeitpunkt der TU wiesen die Sauen überwiegend Corpora lutea (CL; 56,3 %; p < 0,05) auf. Der zweithäufigste Befund waren Follikel von 2-6 mm Durchmesser (F2-6; 27,7 %; p < 0,05). Durch Altrenogest konnte das Follikelwachstum effektiv gehemmt werden, ohne die spontane Luteolyse zu beeinträchtigen. Mehr als 84 % der Tiere wiesen am Ende der 15-tägigen Behandlung mit Regumate® F2-6 auf. Altsauen, die 4 ml Regumate® erhielten, hatten häu-figer als die mit 5 ml behandelten Tiere polyzystisch degenerierte Ovarien (POD). Zahlrei-che Altsauen, vor allem solche mit periovulatorischen Funktionsgebilden zum Zeitpunkt der Trächtigkeitsuntersuchung (p < 0,05), wiesen zudem CL am Ende der Behandlung auf (16,3 %). Jungsauen ovulierten unabhängig von der ovulationsauslösenden Substanz überwiegend zwischen beiden Besamungen (p < 0,05), während Altsauen zu gleichen Anteilen zwischen den Besamungen und nach der zweiten Besamung ovulierten. Die ovulationsauslösende Substanz, d.h. hCG bzw. GnRH, hatte weder bei Jung- noch bei Altsauen einen Einfluss auf das Ovulationsverhalten. Der Ovulationszeitpunkt war ohne Einfluss auf die Trächtig-keitsrate. Altsauen, die D-Phe6-GnRH erhielten, wurden häufiger tragend als Tiere, die hCG erhielten (p < 0,05). Reziprokes Ergebnis erzielten die Jungsauen (p < 0,05). Kon-trolltiere wiesen Trächtigkeits- bzw. Abferkelraten auf, die bis zu 15,6 % (TR; p < 0,05) bzw. 16,8 % (AFR) höher als die der Versuchstiere waren, da vor allem die Altsauen, nicht aber Jungsauen aller drei Versuchsgruppen schlechtere Fruchtbarkeitsleistungen erzielten. Aus den Ergebnissen ist zu schlussfolgern, dass die kombinierte Anwendung von Altrenogest (Regumate®) und Gonadotropinen bzw. GnRH-Analogon D-Phe6-GnRH zur Brunst- und Ovulationssynchronisation bei besamten, als ingravid detektierten Jung- und Altsauen geeignet ist. Ausgehend von den Ergebnissen ist zu empfehlen, Sauen mit 5 ml Reguma-te®/Tier/Tag zu behandeln (Zyklusblockade). Ein Applikationsintervall von 15 Tagen ist ausreichend. Werden 800 IE PMSG/eCG 24 Stunden nach letztmaliger Regumate®-Applikation verabreicht, kann mit zuverlässiger Stimulation des Follikelwachstums gerechnet werden. Jungsauen sollten 500 IE hCG 78 bis 80 Stunden nach der PMSG/eCG-Gabe erhalten. Bei Altsauen sind dazu 50 µg des GnRH-Analogons D-Phe6-GnRH emp-fehlenswert. Die transkutane Ultrasonografie ist ein geeignetes Verfahren zur Darstellung von Ovarien und ovarieller Funktionskörper.

Targeted radionuclide therapy is becoming a widely used cancer treatment strategy. By radiolabeling receptor-specific peptides, cancer cells overexpressing the receptor can be selectively targeted, and the cytotoxic radionuclide can be delivered to the target cell or tissue for therapeutic or diagnostic purposes. Bombesin analogues have been previously developed and utilized to target the gastrin-releasing peptide receptor (GRPR), a receptor commonly overexpressed in prostate cancer cells. The RM26 analogue derived from the native bombesin is an antagonistic ligand of GRPR and a possible candidate for targeted radiotherapy. Prolonging the half-life of the molecule is an important aspect of developing a new protein therapeutic. Using albumin binding domain (ABD) for this purpose is an emerging strategy in recent years. ABD is able to bind to serum albumin and thus remains in the blood circulation for a long period of time. It is also a scaffold for protein engineering efforts and by coupling receptor-specific ligands to ABD, the target-specific binding along with extended in vivo halflife can be achieved. In this project, an RM26 analogue with a PEG linker and ABD with a DOTA chelator for future radiolabeling were synthesized with solid phase peptide synthesis (SPPS), conjugated, purified by RP-HPLC and analyzed by mass spectrometry. The binding properties of the conjugate were evaluated by SPR-based biosensory studies, and further experiments are planned for the testing the product and its potential application in radionuclide therapy. / Riktad radioterapi är en allt vanligare metod för behandling av cancer. Genom att radioinmärka receptor-specifika peptider kan dessa selektivt levereras till tumörceller som uttrycker receptorn. Radioterapi kan användas för diagnostik eller terapi, beroende på kopplad radionuklid. Bombesinanaloger har utvecklats och använts för att selektivt binda gastrinfrisättande peptidreceptor (gastrin-releasing peptide receptor, GRPR), en receptor som ofta är överuttryckt i prostatacancer. Bombesinanalogen RM26, som har sitt ursprung från nativt bombesin, är en antagonist till GRPR och kan möjligen användas för riktad radioterapi av prostatacancer. Vid utvecklingen av nya proteinläkemedel är halveringstiden i serum en viktig aspekt. En nyligen utvecklad strategi för att förlänga halveringstiden i serum är fusion av det  tumörspecifika proteinet till en albumin-bindande domän (ABD). ABD binder till albumin, ochsåledes kan fusionsproteinet bevaras i blodcirkulationen under en längre tid. I detta projekt, har både RM26 med en PEG-linker, och ABD med en DOTA kelator syntetiserats med fastfaspeptidsyntes (solid phase peptide synthesis, SPPS). RM26-PEG och DOTA-ABD har därefter konjugerats, renats med RP-HPLC och analyserats med massspektrometri. Bindning till albumin har utvärderats med ytplasmonresonans (surface plasmon resonance, SPR). Vidare studier planeras för att utvärdera peptid-proteinkonjugatet och dess potential för riktad radioterapi.

No description available.

No description available.