Ifitm基因是一群可被干擾素刺激表現的基因，目前已知這些基因參與細胞對病毒的防禦，然而其生化特性及這些基因參與的其他生物功能並不清楚。將這些基因敲除後小鼠會產生過度攝食的行為並會造成肥胖，同時在下視丘中黑皮質素的前驅物POMC的表現量顯著降低，此發現表明破壞Ifitm基因可導致下視丘功能被破壞。然而由於代謝的調控並非只由下視丘控制，Ifitm基因是否直接參與下視丘的功能並不清楚。本研究卽是在探討Ifitm基因是否在下視丘的功能中扮演重要的角色。我們首先透過與下視丘功能有關的行為分析測試是否有其他下視丘功能被破壞的證據。我們發現在IFITM基因剔除公鼠，3-6個月開始出現過度攝食的行為與體重明顯增加；然而IFITM基因剔除母鼠在18個月後才出現同樣的表現型。IFITM基因剔除公鼠，在OFT與FST的行為測試中行動力下降，IFITM基因剔除母鼠卻有較多的活動；在公鼠與母鼠中焦慮均顯著增加。我們進一步測試這些剔除鼠的下視丘組織及神經胜肽的表現，在組織分析中並沒有發現組織結構異常。在神經胜肽的分析中IFITM基因剔除鼠的CRF的表現量增加，與IFITM基因剔除鼠的焦慮行為顯示相同趨勢。POMC表現的減少與CRF表現的上升顯示下視丘的功能紊亂。我們同時發現IFITM基因剔除鼠中微膠細胞數增加，以及位於下視丘的細胞本體的體積增加。另外IFITM基因剔除鼠中的細胞激素TNF-α與IL-1β在周邊血清中的增加表明此剔除鼠的下視丘產生發炎。本研究發現剔除IFITM基因會造成多項下視丘功能受到影響，並發現此突變鼠有下視丘發炎的現象。此下視丘發炎是否為下視丘功能破壞的主因，及IFITM基因剔除鼠的下視丘功能異常的細胞分子機制將會進一步被分析。 / Ifitm genes are a group of interferon-inducible genes, best known for their antiviral roles. Their biological functions other than cellular antivirus and the biochemical properties of these proteins are not well-understand. Previously age-dependent hyperphagia and obesity were reported in IfitmDel mutant mice, in which 5 Ifitm genes were deleted. The levels of pro-opiomelanocortin (POMC) in hypothalamus of these mice were significantly reduced, suggesting that the hyperphagia phenotype of IfitmDel mutants is caused by defective central melanocortin signaling. In this study, we examine whether the hypothalamic dysfunctions are developed in these mutants. Whether hypothalamus associated behaviors are disrupted in IfitmDel mutants was first examined. We discovered that the hyperphagia and obesity phenotype in IfitmDel mutants are gender-dependent. These phenotypes in male mutants are detectable starting from 4-6 months of age, but not detected until one-year old in female mutants. Male mutants reveal the trends of decreased activities, while female mutants exhibit increased locomotor activity. Both gender develop anxiety-like phenotype. The behavioral abnormalities support the hypothesis of hypothalamic dysfunctions. As the Ifitm genes are expressed in various stage during development, the anatomy and the functions of the hypothalamus of these mutants were further tested. The histological analyses show no anatomical defects, neuron count differences and morphological alterations of neurons in IfitmDel mutant brains of both genders. These results indicate that these proteins are rather crucial for maintaining normal physiology in the developed central nervous system (CNS). The expression levels of neuropeptides related to stress response and anxiety or depression status, such as corticotrophin releasing factor (CRF), are altered in mutant hypothalamus. In contrast, Cell count and cell body size of microglia, whereas sera levels of pro-inflammatory cytokines, including TNF-α and IL-1β are elevated in IfitmDel mice, indicating hypothalamic inflammation and microglial activation phenotypes. The cellular mechanisms underlying the hypothalamic dysfunctions and the causative relationship of the hypothalamic inflammation with these phenotypes shall be further explored in the future studies.

Hypothalamic gonadotropin-releasing hormone (GnRH) (GnRH I) is the central regulator of the mammalian reproductive system. Most vertebrates studied also possess a second form of GnRH, GnRH II. GnRH I acts on its cognate G proteincoupled receptor (GPCR) on pituitary gonadotropes and activates Gq/11-mediated signalling pathways to stimulate the biosynthesis and the release of luteinising hormone (LH) and follicle-stimulating hormone (FSH). Both GnRHs have also been suggested to inhibit cellular proliferation, an action which has largely been proposed to be mediated by the coupling of the receptor to Gi/o. However, the range of G proteins activated by the GnRH receptor and the signalling cascades involved in inducing antiproliferation remain controversial. To delineate the G protein coupling selectivity of the mammalian GnRH receptor and to identify the signalling pathways involved in GnRH I-mediated cell growth inhibition, I examined the ability of the receptor to interact with Gq/11, Gi/o and Gs in Gαq/11 knockout MEF cells. My results indicate that the receptor is unable to interact with Gi/o but can signal through Gq/11. Additionally, my data do not support the suggestion of GnRH receptor-Gs interaction. Furthermore, I show that the GnRH Iinduced inhibition of cell growth is dependent on Gq/11, src and extracellular signal regulated kinase (ERK) but is independent of the activity of protein kinase C (PKC), Ca2+, jun-N-terminal kinase (JNK) or P38. Based on these findings and previous research within our group, I propose a mechanism whereby GnRH I may induce antiproliferation. Previous studies from our laboratory suggest that the GnRH receptor can adopt distinct active conformations in response to the binding of GnRH I and GnRH II. These data thus account for our hypothesis of ligand-induced selective signalling (LiSS). Given my previous results, I examined the ability of the GnRH receptor to couple to G12/13. My work indicates that the receptor can directly activate G12/13 and the downstream signalling cascades associated with this G protein family. Indeed, I provide evidence, in several cellular backgrounds, to suggest that GnRH receptor- G12/13-mediated signalling is involved in the regulation of GnRH-induced MAPK activity, SRE-driven gene transcription and cytoskeletal reorganisation. Furthermore, I propose a role for these G proteins in the transcriptional regulation of LHβ and FSHβ. Finally, I confirm previous results from our laboratory indicating that the GnRH receptor may interact with src Tyr kinase and show that GnRH I but not GnRH II may, independently of Gq/11, stimulate the Tyr phosphorylation and thus the activation of this protein. I propose that this differential signalling accounts for the distinct effects of GnRH I and GnRH II on cellular morphology and SREpromoted transcriptional activity. The research presented within this thesis provides evidence to refute published conclusions based on largely circumstantial experimental data, describes novel GnRH receptor signalling pathways and offers support for the concept of LiSS. It may assist in the development of new therapeutic compounds which selectively target one GnRH-mediated signalling pathway while bypassing others.

The effects of external electric fields (EEF) on brain activity in anesthetized rats were studied. The field strengths used, 9 kV/m and 5 kV/m, both D.C. and A.C. (60Hz) were in the range of those measured beneath current overhead transmission lines. Brain activity was monitored from surface electrodes and from electrodes stereotaxically implanted in the posterior-lateral portion of the hypothalamus. It was found that 9 kV/m and 5 kV/m EEF's both D.C. and A.C. brought about statistically significant changes in hypothalamic activity, however, the effects were bi-directional, (i.e. increases and decreases). Only seven of the 60 animals exposed showed changes in the EEG recorded with surface electrodes. The data clearly indicate that (1) anesthetized animals do respond to a change in the external electric field around them, (2) the hypothalamus may contain special electro-receptors that, in turn, may alter various other physiological processes, and (3) the data indicates the need for further research to help government agencies to establish more adequate safety guidelines.

A área incerto-hipotalâmica (IHy) é uma região diencefálica que atua em funções reprodutivas através da presença do hormônio concentrador de melanina (MCH). Nosso objetivo é estudar as conexões da IHy de fêmeas com áreas relacionadas ao controle neuroendócrino reprodutivo, usando traçador anterógrado dextrana amina biotinilada (BDA) e retrógrado Fluorogold (FG). Nesse sentido, destacamos que a IHy, em fêmeas, conecta-se reciprocamente com diversos núcleos hipotalâmicos da região periventricular e pré-óptica, além das áreas hipotalâmica anterior, posterior e lateral. Nosso resultados também sugerem um dimorfismo sexual das projeções da IHy, já que a área pré-óptica medial, o núcleo anteroventral periventrciular e o núcleo arqueado são mais densamente inervadas pelas IHy em fêmeas do que em machos. / The incerto-hypothalamic area (IHy) is a diencephalic region poorly studied which is characterized by two non-colocalized neurochemical groups composed by the A13 dopaminergic group intermingled with predominantly GABAergic cells co-expressing melanin-concentrating hormone (MCH) and/or cocaine and amphetamine regulated transcript (CART). Functional studies suggest that IHy is involved in the neuroendocrine control of female reproduction in specific metabolic states mediated by MCH. In this way, our aim is to study the IHy connections in female rats related to neuroendocrine control of female reproduction using neuronal anterograde Biotin Dextran Amine (BDA) and retrograde Flurogold (FG) tracers. As a result, we found that IHy projections are the lateral septal nucleus, bed nucleus of stria terminalis, paraventricular thalamic nucleus, reuniens thalamic nucleus, medial preoptic area, medial and lateral preoptic nucleus, median preoptic nucleus, periventricular hypothalamic nucleus, anteroventral periventricular nucleus, anterior, posterior and lateral hypothalamic area and precommissural nucleus. The main afferents to the IHy, in females, are the lateral septal nucleus, median preoptic nucleus and paraventricular thalamic nucleus. These results suggest sexually dimorphic projections from the IHy, since IHy more densely innervates neuroendocrine regions in female than in male rats.

A glândula pineal sintetiza o hormônio melatonina à noite e este regula diversos sistemas fisiológicos, adaptando-os às exigências de cada momento do dia. O objetivo deste trabalho foi o de analisar o perfil noturno da síntese de melatonina na glândula pineal de ratos, em diferentes idades, tratados com glutamato monossódico (MSG) no período neonatal. Ratos Wistar, machos e fêmeas, receberam injeções de MSG (4mg/g/dia) ou de solução salina (0,9%) do 2º ao 8º dia pós-natal. Foram avaliados o peso corporal e dos tecidos adiposos, o comprimento naso-anal,o perfil noturno da síntese de melatonina e da atividade da enzima AANAT, o GTT e o ITT. O MSG induziu um aumento no peso dos tecidos adiposos, sem aumento do peso corporal. Não foi observada hiperglicemia, nem intolerância à glicose, mas sim resistência insulínica. A ritmicidade circadiana da melatonina e da AANAT foi preservada, mas houve um aumento de ambos no ZT 15. As alterações na síntese de melatonina parecem decorrer das lesões hipotalâmicas provocadas pelo MSG, pela redução do NPY, aumento da insulina ou da noradrenalina. / The pineal gland synthesizes melatonin exclusively at night. Melatonin is a temporal synchronizer of several physiological functions, adapting the organism to the diurnal environmental changes. The purpose of this work was to analyze the effects of neonatal monosodium glutamate (MSG) administration on the nocturnal profile of melatonin synthesis in the pineal gland. Wistar rats, males and females, were injected neonatally with MSG (4mg/g/day) or saline solution (0.9%) from the 2nd to 8th post-natal day. Body weight and adipose tissue weight, naso-anal length, nocturnal melatonin and AANAT activity profiles, GTT and ITT were analyzed. MSG induced a great increase in adipose depots without increase in body weight. It did not induce hyperglycemia nor glucose intolerance, but induced insulin resistance. Circadian rhythmicity of melatonin synthesis and AANAT activity was not altered, but there was an increase at ZT 15 in both. It seems that melatonin synthesis changes could be related to hypothalamic lesions, through a reduction in NPY or insulin/norepinephrine elevation.

Ethanol exposure during pregnancy is one of the leading causes of preventable birth defects, leading to a range of symptoms collectively known as fetal alcohol spectrum disorder (FASD). More moderate levels of prenatal ethanol exposure (PNE) lead to a range of behavioural deficits including aggression, poor social interaction, poor cognitive performance and increased likelihood of addiction in later life. Current theories suggest that adaptation in the hypothalamic-pituitaryadrenal (HPA) axis and neuroendocrine systems contributes to mood alterations underlying behavioural deficits and vulnerability to addiction. This has led to the suggestion that corticotrophin-releasing factor (CRF) antagonists and glucocorticoid (steroid) inhibitors may be potential therapeutics to address the deficits of PNE and for the treatment of addiction. The zebrafish (Danio rerio) has several advantages over mammalian models, such as low cost of maintenance, short life cycle, easy embryological manipulation and the possibility of large-scale genetic screening. By using this model, our aim is to determine whether developmental ethanol exposure provokes changes in the HPA axis (HPI axis in fish), as it does in mammalian models, therefore opening the possibilities of using zebrafish to elucidate the mechanisms involved, and to test novel therapeutics to alleviate deleterious symptoms. Thus this thesis focuses solely on the effect of developmental ethanol exposure on the functioning of the HPI axis in zebrafish. Stress-reactivity in zebrafish larvae ethanol-treated 1-9 days post 4 fertilisation (dpf) was assessed using thigmotaxis and thigmotaxis following airstress. In both tests, lower stress-related responses were obtained with ethanol treated animals, in that they spent less time at the edges of the apparatus (P<0.01, n=3). They also showed lower total body cortisol (P=0.04, n=14). Larvae also showed the same behaviour pattern two weeks after ethanol exposure, (23dpf) (P=0.04, n=3), again with reduced total cortisol (P=0.03, n=4). HPI-related gene transcription was also assessed in 9dpf ethanol treated zebrafish larvae, by qRT-PCR. Revealing up-regulation of CRH, CRHBP and CRHR2, normalized against β-Actin, Elav1 and Gap43 housekeeping genes. In situ hybridization revealed no spatial changes in CRH, CRH-BP and POMC with animals at the same stage. Behavioural stress-reactivity differences in 6-months old adults that had been exposed developmentally to ethanol were assessed using novel tank diving and thigmotaxis. Both assays indicated a decrease in stress-like behaviour due to early ethanol exposure compared to controls (P<0.05, n=5 both). Finally, cortisol levels were assayed from 9dpf larvae and 6-month-old adults that had been treated with ethanol during early development showed a significant reduction in cortisol output when air-exposed stressed compared to controls (P=0.04, n=5). Conclusion: Early ethanol exposure produced significant changes in cortisol, HPI gene mRNA expression and stress-reactive behaviour in 9dpf animals. Changes in cortisol and behaviour were still detected in 6-months old adults, developmentally treated with ethanol, indicating that early ethanol exposure has permanent effects on the HPI axis. 5 As our data contradicts the findings in mammalian literature where early ethanol exposure increases stress-like behaviour in later life, it is also possible that more permanent effects of PNE in mammals may arise through maternal-offspring interactions, during and post gestation, such as breastfeeding and maternal grooming of the offspring, which are absent in the zebrafish model.

Les écosystèmes marins constituent une cible majeure des changements globaux qui affectent de façon pérenne notre planète.Nous nous sommes intéressés à deux de ces changements susceptibles de menacer le milieu marin : le réchauffement climatique par le biais de l’étude de l’effet du stress thermique sur l’expression de ho-1 et la pollution via l’étude de l’impact des métaux lourds à effets perturbateurs endocriniens (le cadmium et le plomb) sur l’axe hypothalamo-hypophyso-gonado-hépatique (HHGH) chez le loup (Dicentrarchus labrax L.).Nos résultats montrent une réponse importante de ho-1 aux stress thermique et chimique dans le foie. Nous avons également démontré une forte accumulation hépatique du Cd, et à un degré moindre du Pb, accompagnée d’une sur-expression du gène mt codant pour les métallothionéines. Des variations dans l’expression de gènes clés le long de l’axe HHGH (arom b, fshß, arom a…) ont été observées après intoxication par les deux métaux sans pour autant induire des effets physiologiques observables.La question se pose de savoir si la synergie des stress physico-chimiques impacte la dynamique et l’état sanitaire des populations marines. / Marine ecosystems are a major target of global changes that continuously affect our planet. In the present study we investigated two of these changes that may threaten the marine environment: global warming, through the study of the effect of heat stress on ho-1 expression and marine pollution through the study of the impact of heavy metals acting as endocrine disruptors (cadmium and lead) on the hypothalamic-pituitary-gonadal-liver (HHGL) axis in the sea bass (Dicentrarchus labrax L.). Our results show an important response of ho-1 to both thermal and chemical stress in the liver. We also demonstrated a high hepatic accumulation of Cd, and to a lesser extent of Pb. This accumulation was correlated with an overexpression of mt gene coding for metallothionein. Changes in the expression of candidate genes (arom b, fshß, arom a…) along the HHGL axis were observed after metal intoxication but no physiological effects were observed.The question then arises to what extent the synergy of physicochemical stressors impacts the dynamics and the welfare of marine species.

Abstract not available

Includes bibliographical references (leaves 228-257). Aims to determine the impact of maternal undernutrition during late gestation and during the periconceptional and gestational periods on fetal growth, fetal blood pressure and the fetal hypothalamo-pituitary adrenal axis in the sheep.

Low birth weight is associated with an increased risk of impaired glucose tolerance and type 2 diabetes and with signs of increased hypothalamic pituitary adrenal axis activity in later life (1, 2). Low birth usually weight reflects a reduction in fetal growth, which largely depends on an adequate supply of nutrients and oxygen. Variations in supply modify the metabolic and neuroendocrine characteristics of the fetus, which in turn modulate the pattern of functional development as well as growth (3). An adverse fetal environment, evident as low birth weight, is therefore proposed to alter functional development with long term effects for the function and risk of disease in the individual later in life (4, 5). Increased HPAA impairs metabolic homeostasis and could therefore mediate effect of prenatal challenge on later metabolic control (6). It was therefore hypothesised that restriction of fetal growth, increases circulating cortisol and/or alters sensitivity to cortisol, which increases fasting blood glucose, and impairs glucose tolerance in the young adult. Large litter size in the guinea pig is characterised by reduced placental and fetal growth, reduced size at birth and insulin resistance in offspring in later life, providing a suitable model to test this hypothesis. Spontaneous restriction of fetal growth in the guinea pig, evident as small size at birth, was associated with increased salivary cortisol, in both sexes but at different stages of postnatal life. In males, salivary cortisol was increased with small size at birth in early and adult life, but reduced later with ageing. In females however, salivary cortisol was increased in juveniles and in aged adults, possibly reflecting the impact of the oestrus cycle on cortisol production in mature cycling females. Altered activity of the HPGA, which can influence that of the HPAA, has also been reported to be programmed by prenatal restriction. In the guinea pig, salivary testosterone in males increased with age and small size at birth in juveniles, young and aged adults. In females, salivary progesterone increased with age up to 300 days, and decreased with size at birth in the young guinea pig. Although testosterone inhibits HPAA activity, in males, mean salivary cortisol correlated positively with mean salivary testosterone at 100 and 300 days of age. In contrast, progesterone may enhance HPAA activity, and consistent with this, in females, mean salivary progesterone correlated with mean salivary cortisol at 400 days of age. Therefore, salivary testosterone in the male and salivary progesterone in the female guinea pig changes with maturation and has previously reported in this or other species, but small size at birth increases salivary testosterone in males with modest effects in early life in females. This together with the unexpected positive associations of salivary cortisol with testosterone in males, suggests that programming of the HPAA makes little contribution to that of the HPAA as indicated by salivary cortisol. Here we show that low birth weight is associated with increased fasting blood glucose and impaired glucose tolerance in both male and female young adult guinea pigs aged 100 days. Fasting and mean (during IVGTT) plasma cortisol was reduced in low birth weight female adult guinea pigs, and is not vary with size at birth at this age in males. This suggests that circulating cortisol does not contribute to the impaired glycaemia associated with small size at birth in the guinea pig. Glucose tolerance was increasingly impaired in males but not females, as mean plasma cortisol increased. This is consistent with cortisol impairing glycaemia in the guinea pig as in other species, in males at least. To assess the role of cortisol in prentally programmed impairment of glycaemia directly, metyrapone or vehicle containing 24% ethanol was administered to young adult guinea pigs for 3 days. Treatment with the latter impaired fasting blood glucose and glucose tolerance in females and the latter in males compared to a previous IVGTT and this was exacerbated in low birth weight females. Metyrapone prevented this impairment of fasting glycaemia and glucose tolerance in the low birth weight adult female guinea pig and in the male guinea pig regardless of birth weight class. Neither vehicle or metyrapone altered plasma cortisol, before or during a second IVGTT. Limited numbers of animals, particularly females, limited this study however and additional investigation is required. Nevertheless this shows for the first time that inhibition of glucocorticoid synthesis in the guinea pig improves glucose control. Furthermore this suggests that the low birth weight guinea pig may be more sensitive to cortisol, have increased cortisol synthesis or reduced inactivation of cortisol in peripheral tissues, leading to increased local cortisol action. In conclusion, alterations in peripheral HPAA activity in the guinea pig due to restricted fetal growth may contribute to their prenatally programmed development of impaired glucose tolerance as young adults, but the extent of that contribution may vary with age and gender. / Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2008