Prader-Willi syndrome (PWS) is a rare, neurodevelopmental disorder with multiple features caused by hypothalamic deficiency, including infantile failure to thrive, hyperphagia leading to obesity, growth hormone deficiency, hypogonadism, and central adrenal insufficiency. Other features of PWS including global developmental delay, hypotonia, pain insensitivity, gastrointestinal dysfunction, and psychiatric disorders are caused by deficits in other regions of the nervous system. PWS is caused by the loss of a subset of paternally-expressed genes on chromosome 15, which includes NDN and MAGEL2. Necdin and Magel2 are both members of the melanoma antigen (MAGE) family of proteins and are expressed throughout development, particularly in the nervous system. This thesis describes experiments that examine the loss of function of necdin and Magel2 in mice and their potential roles in the pathogenesis of PWS. Targeted inactivation of Ndn and Magel2 in mice has aided in determining how loss of function of these proteins affects the development and function of the nervous system. Loss of necdin causes reduced axonal outgrowth and neuronal differentiation in the central and peripheral sensory nervous systems. I examined the autonomic nervous system in Ndn-null embryos and identified a defect in the migration of the most rostral sympathetic chain ganglion and consequently increased neuronal cell death and reduced innervation of target tissues supplied by this ganglion. Reduced axonal outgrowth was observed throughout the sympathetic nervous system in Ndn-null embryos although no gross deficits in the parasympathetic and enteric nervous systems were identified. Loss of Magel2 causes reduced fertility and abnormal circadian rhythm patterns in mice. I further identified an altered response to stress, a delayed response to insulin-induced hypoglycemia, a reduced stimulated growth hormone response, and lower thyroid hormone levels in Magel2-null mice, indicative of deficits in multiple hypothalamic-pituitary axes. The findings presented in this thesis support a role for necdin and Magel2 in the development and function of the nervous system. The data also indicates that these MAGE proteins play a key role in multiple features of PWS, including endocrine deficiencies and autonomic dysfunction

Object: The aim of the study was to evaluate the early changes in pituitary hormone levels after severe traumatic brain injury (sTBI) and compare hormone levels to basic neuro-intensive care data, a systematic scoring of the CT-findings and to evaluate whether hormone changes are related to outcome. Methods: Prospective study, including consecutive patients, 15-70 years, with sTBI, Glasgow Coma Scale (GCS) score <= 8, initial cerebral perfusion pressure > 10 mm Hg, and arrival to our level one trauma university hospital within 24 hours after head trauma (n = 48). Serum samples were collected in the morning (08-10 am) day 1 and day 4 after sTBI for analysis of cortisol, growth hormone (GH), prolactin, insulin-like growth factor 1 (IGF-1), thyroid-stimulating hormone (TSH), free triiodothyronine (fT3), free thyroxine (fT4), follicular stimulating hormone (FSH), luteinizing hormone (LH), testosterone and sex hormone-binding globulin (SHBG) (men). Serum for cortisol and GH was also obtained in the evening (17-19 pm) at day 1 and day 4. The first CT of the brain was classified according to Marshall. Independent staff evaluated outcome at 3 months using GOS-E. Results: Profound changes were found for most pituitary-dependent hormones in the acute phase after sTBI, i.e. low levels of thyroid hormones, strong suppression of the pituitary-gonadal axis and increased levels of prolactin. The main findings of this study were: 1) A large proportion (54% day 1 and 70% day 4) of the patients showed morning s-cortisol levels below the proposed cut-off levels for critical illness related corticosteroid insufficiency (CIRCI), i.e. < 276 nmol/L (= 10 ug/dL), 2) Low s-cortisol was not associated with higher mortality or worse outcome at 3 months, 3) There was a significant association between early (day 1) and strong suppression of the pituitary-gonadal axis and improved survival and favorable functional outcome 3 months after sTBI, 4) Significantly lower levels of fT3 and TSH at day 4 in patients with a poor outcome at 3 months. 5) A higher Marshall CT score was associated with higher day 1 LH/FSH-and lower day 4 TSH levels 6) In general no significant correlation between GCS, ICP or CPP and hormone levels were detected. Only ICPmax and LH day 1 in men was significantly correlated. Conclusion: Profound dynamic changes in hormone levels are found in the acute phase of sTBI. This is consistent with previous findings in different groups of critically ill patients, most of which are likely to be attributed to physiological adaptation to acute illness. Low cortisol levels were a common finding, and not associated with unfavorable outcome. A retained ability to a dynamic hormonal response, i.e. fast and strong suppression of the pituitary-gonadal axis (day 1) and ability to restore activity in the pituitary-thyroid axis (day 4) was associated with less severe injury according to CT-findings and favorable outcome.

Following social defeat, male hamsters exhibit behavioral changes characterized by a breakdown of normal territorial aggression and an increase in submissive/defensive behaviors in the presence of a non-aggressive intruder (NAI). We have termed this phenomenon conditioned defeat (CD). By contrast, only a small subset of defeated females exhibit submissive/defensive behavior in the presence of a NAI. We hypothesized that fluctuations in gonadal hormones might contribute to differences in the display of submissive behavior in intact female hamsters. Following social defeat, proestrous females (higher endogenous estradiol) were more likely to display conditioned defeat compared with diestrous 1 (lower endogenous estradiol) females. This finding suggests that there is an estrous cycle-dependent fluctuation in the display of CD in female hamsters and suggests that increased estradiol might contribute to increased submissive behavior. We then demonstrated that ovariectomized females given estradiol prior to CD testing exhibited significantly higher submissive behavior in the presence of a NAI suggesting that estradiol increases the expression of CD in female hamsters. We have also shown that castrated males that were singly housed for four weeks displayed significantly more submissive behavior than did their intact counterparts. Interestingly, castrated and intact males that were singly housed for 10 days prior to behavioral testing displayed similar behavior during CD testing. Together these data suggest that androgens and isolation modulate the display of CD in male hamsters. Finally, we examined brain activation following CD testing in defeated males and females (in diestrus 1 and proestrus). Defeated male and proestrous females exhibited increased Fos activation in the dorsal lateral septum and hypothalamic paraventricular nucleus relative to defeated diestrous 1 females. Diestrous 1 females exhibited increased Fos expression in the lateral bed nucleus of the stria terminalis compared with both defeated groups. Collectively, these data suggest that gonadal hormones and duration of individual housing modulate the display of CD in female and male hamsters and that those animals which display CD exhibit differences in patterns of neuronal activation than do those that do not display CD.

published_or_final_version / Medicine / Master / Doctor of Medicine

No description available.

No description available.

This project investigated postnatal development of the adrenocortical function in captive American kestrels (Falco sparverius) employing measurements of basal and stress-induced levels of corticosterone at specific developmental stages. Chicks aged 10-days exhibited partially functioning adrenocortical systems with baseline levels comparable to adults. The ability to respond to external stressors increased through postnatal development and by the age of 22 days, stress-induced maximal levels of corticosterone were indistinguishable from those of one-year old adults, and levels of 28-day old birds were significantly higher than these adults. In addition, baseline and maximum stress-induced levels of corticosterone at all ages were significantly higher in first-hatched chicks than all other siblings and these effects grew stronger through development. These results suggest that the brain-pituitary-adrenal axis in this semi-altricial species is (1) already partially developed in young chicks and (2) only becomes fully functional when behavioral and neuromuscular development is nearly complete. Furthermore, results from this study suggest that hatching asynchrony has an effect on this variation in stress-induced maximal levels of corticosterone during the latter half of postnatal development, with a higher degree of hatching asynchrony leading to larger disparity in adrenocortical function between first- and fourth-hatched chicks. This adrenocortical disparity resulting from female-mediated hatching asynchrony may potentially lead to both brood-reduction and brood survival under diametric food conditions, ensuring that the female's reproductive fitness is maximized in varying habitats. Variation of adrenocortical function among siblings may increase female efficiency in raising a brood of fit chicks, maximizing her reproductive success.

Arginine vasopressin (AVP) is a hypothalamic nonapeptide which regulates the hypothalamic-pituitary-adrenal axis response to stress by stimulating the secretion of adrenocorticotropin (ACTH) from corticotroph cells of the anterior pituitary. This effect is mediated by binding of AVP to the pituitary vasopressin receptor (V1bR). The V1bR belongs to the G protein-coupled receptor (GPCR) super family. Repeated stimulation of anterior pituitary cells with AVP has been shown to produce a loss of responsiveness to subsequent AVP stimulation. This phenomenon appears to be mediated by desensitisation of the V1bR, and may be due to phosphorylation of the receptor by G protein-coupled receptor kinase 5 (GRK5). The aim of this research was to establish and validate methods that would allow the role of GRK5 in the desensitisation of V1bR to AVP stimulation to be investigated. As no isoform specific inhibitors for GRK5 were available, HEK293 cells transiently transfected with the rat V1bR were used as a model system for this research. This allowed RNA interference (RNAi) to be used to knockdown GRK5 expression. The protocol for RNAi-mediated knockdown of GRK5 was established as part of this research. Protocols for Western blotting and qRT-PCR were also established to allow the RNAi-mediated knockdown of GRK5 protein and mRNA to be measured. Transfection of HEK293 cells with 10nM GRK5-targeting small interfering RNAs (siRNAs) reduced the expression of GRK5 protein to 53.4% ± 3.4% (mean ± SEM) of that seen in untreated control cells at 84 hours after transfection, while GRK5 mRNA levels were reduced to 28.7% ± 1.9% (mean ± SEM) of that of control cells 48 hours after transfection. An experimental protocol was designed in this research that would coordinate the RNAi-mediated knockdown of GRK5 with transient transfection of the HEK293 cells with the rV1bR. Since, activated V1bRs couple to Gq/11 and stimulate the production of inositol phosphates (IPs), the responsiveness of the V1bR can be determined by measuring the accumulation of [H³]-IPs in cells labelled with [H³]-myo-inositol. In the protocol designed, the effect of GRK5 knockdown on V1bR desensitisation is determined by stimulating HEK293 cells expressing the rV1bR (and previously transfected with GRK5-targeting siRNA) with 0nM or 100nM AVP for 0, 5, 15, 30 or 60 minutes, and comparing the accumulation if IPs over time with that of cells that are not transfected with GRK5-targeting siRNA. This protocol can be used in future to investigate the role of GRK5 in V1bR desensitisation, and may be adapted to determine if other GRK isoforms are involved in V1bR desensitisation.

Chronic major depressive disorder (CMDD) is a common and debilitating illness. Its pathophysiology needs further elucidation, before more effective targeted treatments can be developed for this condition. To gain a better understanding of the psychobiology of CMDD, three interconnected studies were conducted that examined the interplay between chronic depression, cortisol responses, and personality. Study 1 examined cortisol responses to the Trier Social Stress Test (TSST) in CMDD participants (n=29) as compared to healthy controls (n=28). It was hypothesized that cortisol responses would be greater in the CMDD population. Results indicated that females with CMDD had increased cortisol output compared to female controls, a pattern consistent with the hypothesis. However, males with CMDD had decreased cortisol responses compared to male controls. These results suggest that cortisol responses to social stress are altered in those with CMDD; however, females and males experience fundamentally different changes. Study 2 examined moderating effects of personality on cortisol responses to the TSST in those with CMDD (n=51) as compared to healthy controls (n=57). It was hypothesized that higher neuroticism and/or lower extraversion would be associated with increased cortisol responses in CMDD participants. As hypothesized, lower extraversion was associated with increased cortisol reactivity in those with CMDD but not in healthy controls. However, no association was found between neuroticism and cortisol responses. These findings could support the notion that lower extraversion is a vulnerability marker for chronic depression and thus a possible target for treatment. Study 3, evaluated the cortisol awakening response (CAR) in CMDD participants (n=27) compared to healthy controls (n=30). It was hypothesized that such awakening responses would be more pronounced in the depressed population compared to controls. Contrary to expectation, no differences were found between the groups. However, lower extraversion was associated with a lower CAR in both CMDD and healthy controls, a finding that was not anticipated a priori. These interconnected studies suggest that examining relationships between depression, cortisol responses, and personality, can assist with identifying distinct psychobiological profiles in those with chronic depression. It is proposed that this strategy will improve the likelihood of developing more targeted treatments for this population.

Chronic major depressive disorder (CMDD) is a common and debilitating illness. Its pathophysiology needs further elucidation, before more effective targeted treatments can be developed for this condition. To gain a better understanding of the psychobiology of CMDD, three interconnected studies were conducted that examined the interplay between chronic depression, cortisol responses, and personality. Study 1 examined cortisol responses to the Trier Social Stress Test (TSST) in CMDD participants (n=29) as compared to healthy controls (n=28). It was hypothesized that cortisol responses would be greater in the CMDD population. Results indicated that females with CMDD had increased cortisol output compared to female controls, a pattern consistent with the hypothesis. However, males with CMDD had decreased cortisol responses compared to male controls. These results suggest that cortisol responses to social stress are altered in those with CMDD; however, females and males experience fundamentally different changes. Study 2 examined moderating effects of personality on cortisol responses to the TSST in those with CMDD (n=51) as compared to healthy controls (n=57). It was hypothesized that higher neuroticism and/or lower extraversion would be associated with increased cortisol responses in CMDD participants. As hypothesized, lower extraversion was associated with increased cortisol reactivity in those with CMDD but not in healthy controls. However, no association was found between neuroticism and cortisol responses. These findings could support the notion that lower extraversion is a vulnerability marker for chronic depression and thus a possible target for treatment. Study 3, evaluated the cortisol awakening response (CAR) in CMDD participants (n=27) compared to healthy controls (n=30). It was hypothesized that such awakening responses would be more pronounced in the depressed population compared to controls. Contrary to expectation, no differences were found between the groups. However, lower extraversion was associated with a lower CAR in both CMDD and healthy controls, a finding that was not anticipated a priori. These interconnected studies suggest that examining relationships between depression, cortisol responses, and personality, can assist with identifying distinct psychobiological profiles in those with chronic depression. It is proposed that this strategy will improve the likelihood of developing more targeted treatments for this population.