Thesis advisor: Charles S. Hoffman / The biological roles of the most recently discovered mammalian cyclic nucleotide phosphodiesterase (PDE) family, PDE11, are poorly understood, in part due to the lack of selective inhibitors. To address this need for such compounds I completed a ~200,000 compound high throughput screen (HTS) for PDE11 inhibitors using a yeast-based growth assay. Further characterization of lead candidates using both growth-based assays in the fission yeast Schizosaccharomyces pombe and in vitro enzyme assays identified four potent and selective PDE11 inhibitors. I examined the effect of these compounds on human adrenocortical cells, where PDE11 is believed to regulate cortisol levels. One compound, along with two structural analogs, elevates cAMP levels and cortisol production through PDE11 inhibition, thus phenocopying the behavior of adrenocortical tumors associated with Cushing syndrome. These compounds can be used as research tools to study the biological function of PDE11, and can also serve as leads to develop therapeutic compounds for the treatment of adrenal insufficiencies. This study further validates the yeast-based HTS platform as a powerful tool for the discovery of potent, selective and biologically-active PDE inhibitors. / Thesis (PhD) — Boston College, 2012. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Introdução: A exposição frequente dos profissionais de enfermagem a estressores relacionados ao trabalho tem sido amplamente descrita na literatura. Entretanto, a magnitude e intensidade do estresse dependem não somente dos estressores, mas também da interação destes com a avaliação cognitiva da situação estressora, com os recursos de enfrentamento e com a reação psiconeuroendócrina do estresse. Embora diversos estudos tenham descrito as reações psicológicas do estresse e seu enfrentamento nos profissionais de enfermagem, pouco se sabe sobre as características da reação neuroendócrina. Isto é particularmente importante, dado que padrões atípicos de secreção diurna de cortisol, principal hormônio do estresse, estão associados ao aumento da susceptibilidade ao desenvolvimento de doenças cardiovasculares, imunológicas e transtornos mentais. Assim, questiona-se a frequência de padrões atípicos de cortisol e sua relação com manifestações psicológicas nestes profissionais. Objetivo: Analisar o padrão diurno de secreção de cortisol dos profissionais de enfermagem de unidades hospitalares. Método: Foram incluídos 56 profissionais de enfermagem randomicamente selecionados, alocados nas unidades ambulatório, clínica médica, clínica cirúrgica, centro cirúrgico, pronto socorro infantil e adulto, unidade de terapia intensiva adulto e pediátrica de um hospital universitário. Para avaliação do padrão diurno de secreção de cortisol foram coletadas amostras de saliva em dois dias úteis consecutivos de trabalho e, para as manifestações psicológicas, foram aplicados os instrumentos escala de estresse percebido (EEP), questionário de sofrimento mental (SRQ-20), inventário de depressão de Beck (IDB) e escala de estresse no trabalho (EET). Os dados foram armazenados e analisados utilizando o programa estatístico SPSS versão 14.0 e o nível de significância adotado foi de 5%. Resultados: Quanto ao padrão de secreção de cortisol, 42,5% dos profissionais de enfermagem apresentaram padrão atípico de secreção de cortisol, sendo que 19,5% eram técnicos de enfermagem. Quanto às variáveis psicológicas, 54,5% perceberam-se com alto nível de estresse (EEP), 51,2% referiram que o estresse estava relacionado ao trabalho (EET), 15,5% apresentaram distmia e depressão (IDB) e 56,8% apresentam sinais de sofrimento mental (SRQ-20). Não houve associação entre padrão de secreção de cortisol e as variáveis psicológicas. Conclusão: Mais de um terço da amostra de profissionais de enfermagem apresentou padrões atípicos de secreção de cortisol, além de relatarem elevados níveis de estresse, estresse relacionado ao trabalho e sofrimento mental. Estes dados sugerem que estes profissionais podem estar expostos a uma sobrecarga não apenas mental, mas biológica, estando expostos ao risco para o adoecimento. / Background: Frequent exposure of nurses to work-related stressors has been widely described in the literature. However, the magnitude and intensity of the stress depends not only on stressors, but also their interaction with the cognitive appraisal of stressful situation, the resources and coping with the psychoneuroendocrine stress response. Although several studies have described the psychological reactions of stress and coping with it in nursing, little is known about the characteristics of the neuroendocrine response of stress. This is particularly important since atypical patterns of diurnal cortisol secretion are associated with increased susceptibility to the development of cardiovascular, immunological diseases and mental disorders. Thus, we arised the question about the frequency of atypical patterns of cortisol and its relationship with psychological manifestations in nursing professionals. Objective: To analyze the diurnal pattern of cortisol secretion of nursing professionals at a hospital setting. Methods: Fifty six (n = 56) nursing professionals were randomly selected allocated to the outpatient clinic, medical clinic, surgical clinic, surgical center, pediatric unit, adult and pediatric emergency department and intensive care unit of a university hospital. To evaluate the diurnal pattern of cortisol secretion, saliva samples were collected on two consecutive working days. For the psychological manifestations of stress the following instruments were applied: the Perceived Stress Scale (PSS), the Self-report Questionnaire (SRQ-20), the Beck Depression Inventory (BDI) and the work-related stress scale (WSS). Data were stored and analyzed using SPSS version 14.0 and the level of significance was 5%. Results: Regarding the pattern of cortisol secretion 42.5% of nurses had atypical pattern of cortisol secretion and 19.2% were from nursing technical professional category. Regarding psychological variables, 54.5% perceived themselves at high stress level, 51.2% reported that stress were work-related 15.5% had depression and dysthymia and 56.8% showed signs of mental distress. There was no association between pattern of cortisol secretion and the psycological variables. Conclusion: More than one third of the sample exhibited atypical pattern of cortisol secretion as well as high levels of stress, work-related stress and mental distress. These data suggest that these workers may be exposed not just to psychological overload, but also to biological burden and could be exposed to a risk for the illness.

Dirección General de Investigación Científica y Técnica (PB 87-0093)

by Linda Shiou-mei Ooi. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 95-109). / Abstract --- p.i / Acknowledgements --- p.iii / List of Figures --- p.viii / Chapter Chapter I. --- Introduction --- p.1 / Objectives --- p.4 / Chapter Chapter II. --- Literature Review --- p.5 / Chapter II. 1. --- Lipoprotein-Lipids --- p.5 / Chapter II.1.1. --- General Concept and Metabolism of Lipoprotein-lipids --- p.5 / Chapter II. 1.2. --- Factors affecting plasma lipoprotein-lipids --- p.11 / Chapter II. 1.2.1. --- Ageing --- p.11 / Chapter II. 1.2.2. --- Obesity --- p.12 / Chapter II. 1.2.3. --- Diet --- p.13 / Chapter II. 1.2.4. --- Alcohol --- p.13 / Chapter II. 1.2.5. --- Cigarette smoking --- p.14 / Chapter II. 1.2.6. --- Exercise --- p.14 / Chapter II. 1.2.7. --- Gender differences --- p.14 / Chapter II.2. --- Sex Hormones --- p.14 / Chapter II.2.1. --- General concepts of sex hormone-production and the metabolism of sex hormones --- p.15 / Chapter II.2.1.1. --- Biosynthesis of testosterone --- p.16 / Chapter II.2.1.2. --- Metabolism of testosterone --- p.17 / Chapter II.2.1.3. --- Dihydrotestosterone (DHT) --- p.18 / Chapter II.2.1.4. --- Androstenedione --- p.18 / Chapter II.2.1.5. --- Biosynthesis of estrogen in men --- p.18 / Chapter II.2.1.6. --- Metabolism of estrogen --- p.19 / Chapter II.2.2. --- Factors affecting sex hormone levels in plasma --- p.19 / Chapter II.2.2.1. --- Sex hormone binding globulin (SHBG) --- p.19 / Chapter II.2.2.2. --- Sampling time --- p.19 / Chapter II.2.2.3. --- Stress and acute or chronic non-endocrine illnesses --- p.20 / Chapter II.2.2.4. --- Ageing --- p.21 / Chapter II.2.2.5. --- Diet and nutrition --- p.21 / Chapter II.2.2.6. --- "Medication, drugs and alcohol" --- p.22 / Chapter II.2.2.7. --- Body composition and obesity --- p.22 / Chapter II.2.2.8. --- Variations in states of sleep-wake cycle --- p.23 / Chapter II.2.2.9. --- Levels of physical and sympathetic nervous system activity --- p.23 / Chapter II. 3. --- The relationship between sex hormones and lipoproteins --- p.24 / Chapter II.3.1. --- "Gender difference in sex hormones, menopause and lipoprotein-lipids" --- p.24 / Chapter II.3.2. --- "Interventional study, exogenous sex hormone and lipoprotein-lipidsin men" --- p.25 / Chapter II.3.3. --- The relationships of endogenous sex hormones and lipoprotein-lipids --- p.26 / Chapter Chapter III. --- Materials and Methods --- p.28 / Chapter III.l. --- Subjects and Sampling Methods --- p.28 / Chapter III.2. --- Quantitation of serum lipoprotein-lipids --- p.29 / Chapter III.2.1 --- Determination of cholesterol and triglyceride --- p.29 / Chapter Table III.2.1.A. --- Intra-assay variations for cholesterol and triglyceride --- p.30 / Chapter Table III.2.1.B. --- Inter -assay variations for Cholesterol and Triglyceride --- p.30 / Chapter III.2.2. --- Determination of HDL-Cholesterol and its subfractions --- p.31 / Chapter Table III.2.2. --- Intra- and inter-assay variation for HDL-cholesterol --- p.31 / Chapter III.2.3. --- Determination of VLDL-C and LDL-C --- p.32 / Chapter III.2.4. --- Quantitative determination of serum apolipoproteins and Lp(a) --- p.32 / Chapter III.2.4.1. --- Determination of Apolipoproteins A-I and B --- p.33 / Chapter III.2.4.2. --- Determination of Lipoprotein (a) --- p.33 / Chapter III. 3. --- Quantitative determination of sex hormones --- p.33 / Chapter III.3.1. --- For urinary unconjugated and serum total testosterone --- p.34 / Chapter III.3.1.1 --- Experimental Procedures --- p.35 / Determination of the optimal antibody titre --- p.35 / Establishment of a standard curve and quality controls --- p.35 / Preparation of standards --- p.35 / Purification of radioactively-labelled 3H-testosterone --- p.37 / Preparation of charcoal- stripped urine as zero calibrator (blank) --- p.37 / Preparation of spiked urine or plasma --- p.38 / Preparation of samples for RIA --- p.38 / RIA --- p.39 / Calculation --- p.40 / Chapter III.3.1.2. --- Characteristics of the radioimmunoassay for testosterone --- p.40 / Sensitivity --- p.40 / Precision studies --- p.42 / Within- and between- batch imprecisions --- p.42 / Chapter Table III.3.1.2.A. --- Within-run variation --- p.42 / Chapter Table III.3.1.2.B. --- Between-run variation --- p.42 / Recoveries --- p.43 / Chapter Table III.3.1.2.C. --- "The recoveries of known amounts of testosterone added to charcoal-stripped urine, between immunoassays" --- p.43 / Test of linearity --- p.43 / Comparison with another procedure --- p.43 / Cross reactivity of the antiserum --- p.44 / Procedure --- p.46 / Chapter Table III.3.1.2.D. --- Cross reactivity of some naturally occurring steroids with testosterone antiserum --- p.47 / Chapter III.3.2 --- For urinary total testosterone --- p.47 / Test of linearity and recovery --- p.48 / Chapter III.3.3. --- For urinary unconjugated and serum total 17β-Estradiol --- p.50 / Chapter III.3.3.1 --- Experimental procedure --- p.50 / Determination of the optimal antibody titre --- p.50 / Establishment of a standard curve and quality controls --- p.52 / Preparation of standards --- p.52 / Preparation of tracer 3H-estradiol and construction of a standard curve --- p.52 / Preparation of spiked control urine or plasma --- p.52 / Preparation of samples for RIA --- p.53 / Chapter III.3.3.2. --- Characteristics of the radioimmunoassay for E2 --- p.53 / Sensitivity --- p.54 / Precision studies --- p.54 / Within- and between- batch imprecisions --- p.54 / Chapter Table III.3.3.2.A. --- Within-run variation of known amount of E2 added to charcoal- stripped urine --- p.54 / Chapter Table III.3.3.2.B. --- Between-run variation of known amount of e2 added to charcoal- stripped urine --- p.54 / Recoveries --- p.56 / Chapter Table III.3.3.2.C. --- "The recoveries of known amount of e2 added to charcoal- stripped urine, between immunoassays" --- p.56 / Test of linearity --- p.56 / Comparison with another procedure --- p.56 / Chapter III.3.4. --- For urinary total estradiol --- p.58 / Test of linearity and recovery --- p.58 / Chapter III.4. --- Determination of serum sex hormone-binding globulin (SHBG) --- p.60 / Chapter III.5. --- Determination of urinary unconjugated Cortisol --- p.60 / Chapter III.6. --- Statistical methods --- p.62 / Chapter III.6.1. --- Biological Variations --- p.62 / Chapter III.6.2. --- Univariate and multivariate correlations --- p.62 / Chapter Chapter IV. --- Results --- p.64 / Chapter IV. 1. --- The characteristics of the experimental subjects and their lipoprotein-lipids profiles --- p.64 / Chapter Table IV. 1. A. --- The anthropometric and biochemical characteristics of the experimental male subjects --- p.64 / Chapter Table IV.1.B. --- The lipoprotein-lipids profiles in 46 healthy Hong Kong Chinese men --- p.65 / Chapter IV. 2. --- "Levels of sex hormones in serum and urine, and urinary free Cortisol" --- p.65 / Chapter Table IV.2. --- The sex hormones at serum and urinary levels and urinary free Cortisol in 46 healthy Hong Kong Chinese men --- p.66 / Chapter Table IV.2.A. --- Formula for the indirect calculation of unbound (free) testosterone levels in plasma --- p.67 / Chapter Table IV.2.B. --- Formula for the indirect calculation of unbound (free) 17β-estradiol levels in plasma --- p.68 / Chapter IV. 3. --- Biological variations --- p.69 / Chapter Table IV. 3. --- "The biological variations of serum lipoprotein-lipids, serum sex hormones and urinary sex hormones and Cortisol in 46 healthy Hong Kong Chinese men" --- p.69 / Chapter Table IV.3.A. --- Correlations of serum lipoprotein-lipids between short-term (3- week) variations --- p.70 / Chapter Table IV.3.B. --- Correlations of serum and urinary sex hormones and urinary unconjugated Cortisol between short-term (3-week) variations --- p.70 / Chapter IV. 4. --- Univariate correlation --- p.71 / Chapter Table IV.4. --- The univariate correlation table --- p.72 / Chapter IV.4.1. --- Inter-relationship among serum and urinary sex hormones --- p.73 / Chapter IV.4.2. --- Urinary free Cortisol and sex hormones and serum lipoprotein-lipids --- p.73 / Chapter IV.4.3. --- Correlation between urinary sex hormones and serum lipoprotein- lipids --- p.73 / Chapter IV.4.4. --- Correlations among serum lipoprotein-lipids --- p.74 / Chapter IV.4.5. --- Correlations between serum lipoprotein-lipids and sex hormones --- p.74 / Chapter IV.4.6. --- "Correlations between anthropometric variables, sex hormones and lipoprotein-lipids" --- p.75 / Chapter IV.4.7. --- Correlation of the ratio of HDL2 and HDL3 and other variables --- p.76 / Chapter IV. 5. --- Multiple linear stepwise regression --- p.77 / Chapter Table IV.5. --- "Stepwise multiple linear regression of lipoprotein-lipids on BMI, W/H Ratio, and Age" --- p.77 / Chapter Table IV.5. A. --- "Stepwise multiple linear regression of lipoprotein-lipids on BMI, W/H Ratio, Age, SHBG, and serum and urinary Sex Hormones" --- p.80 / Chapter Table IV.5.B. --- "Stepwise multiple linear regression of lipoprotein-lipids on BMI, W/H Ratio, Age, SHBG, Triglyceride and serum and urinary Sex Hormones" --- p.81 / Chapter Chapter V. --- Discussion --- p.82 / Chapter V. l. --- Experimental subjects and their lipoprotein-lipids profiles --- p.82 / Chapter V. 2. --- Levels of sex hormones in serum and urine --- p.84 / Chapter Table V.2. --- Values of sex steroids in 46 healthy Hong Kong Chinese men compared to others cited in literature --- p.85 / Chapter V. 3. --- "17β-Estradiol,atherogenic lipoprotein-lpids and HDL3" --- p.88 / Chapter V. 4. --- "Testosterone, and HDL-C and its subfractions" --- p.90 / Chapter Chapter VI. --- Conclusions --- p.92 / References --- p.95 / Appendices --- p.110

Thesis (M.A.)--State University of New York at Binghamton, Psychology Department, 2006. / Includes bibliographical references.

Estímuls estressants bàsicament emocionals, com la immobilització en taula (IMO), exerceixen efectes a llarg termini sobre l'activitat de l'eix hipotalàmic-pituïtari-adrenal (HPA) en rates adultes. Aquests efectes es caracteritzen per una desensibilització, primer a nivell perifèric (d'adrenocorticotropina (ACTH) y de corticosterona al cap d'uns dies) i més tard a nivell central (d'mRNA de corticoliberina (CRF) en el nucli paraventricular de l'hipotàlem (PVN) al cap d'unes setmanes), de la resposta d'aquest eix endocrí quan s'exposen els animals per segona vegada al mateix estrès. Aquest fenomen presenta força similituds amb l'anomenada síndrome d'estrès post-traumàtic en humans, i podria emmarcar-se dins del concepte general de plasticitat sinàptica.Les preguntes que es pretengueren respondre amb aquest treball foren les següents: (i) es poden generalitzar aquests efectes a un estrès físic-immune com l'administració de lipopolisacàrid bacterià (LPS)?, (ii) és el fenomen específic de l'estímul aplicat?, (iii) té el fenomen un correlat en l'activitat transcripcional primerenca en el PVN?, i (iv) quines són les àrees cerebrals sensibles a una experiència prèvia amb estrès?.S'utilitzaren rates mascle adultes de la soca Sprague-Dawley, que s'estressaren només dues vegades per IMO o LPS, separades en el temps entre 1 i 4 setmanes. S'analitzaren en plasma ACTH i corticosterona per radioimmunoanàlisi, i factor de necrosi tumoral (TNF)-a per ELISA. Es van mesurar els nivells d'mRNA de c-fos, CRF i receptor de glucocorticoides (GR) i d'hnRNA de CRF i vasopressina (AVP) per hibridació in situ.L'LPS reduí la resposta de l'mRNA de CRF en el PVN al mateix estímul 4, però no 2, setmanes més tard; la resposta perifèrica, així com la del TNF-a, també fou menor, tant 1 com 4 setmanes més tard. L'LPS bloquejà la hipotèrmia per LPS 4 setmanes més tard. No s'observà desensibilització creuada entre IMO i LPS en cap dels paràmetres de l'eix HPA estudiats. La IMO prèvia reduí la resposta de l'mRNA de c-fos a la IMO en el PVN, tant 1 com 4 setmanes més tard, així com la de l'hnRNA de CRF només a les 4 setmanes. S'observaren efectes similars amb LPS a nivell de l'mRNA de c-fos, però no d'hnRNA de CRF, a les 4 setmanes. La IMO o l'LPS previs no van alterar la resposta de l'hnRNA d'AVP al mateix estímul. En general, no s'observaren efectes creuats consistents entre LPS i IMO a nivell d'hnRNA de CRF o de AVP, o d'mRNA de c-fos en el PVN. La IMO o l'LPS no van modificar els nivells basals d'mRNA de GR a la formació hipocampal i al PVN 4 setmanes després.El patró d'activació cerebral de l'mRNA de c-fos per IMO o LPS fou diferent. Les àrees cerebrals sensibles a la IMO prèvia foren la divisió ventral del septum lateral, el PVN, l'amígdala medial, la divisió medial ventral del nucli de l'estria terminal (BST), el locus coeruleus (LC) i el nucli del tracte solitari. En el cas de l'LPS, aquestes àrees foren el PVN, l'amígdala central, la divisió lateral del BST i el LC.Dels resultats obtinguts es desprèn que els efectes a llarg termini de l'estrès es poden generalitzar a estímuls de naturalesa física-immune com l'LPS. A més, el fenomen sembla ser específic de l'estímul aplicat i es detecta també en l'activitat transcripcional primerenca en el PVN. Finalment, postulem que les estructures cerebrals més probablement responsables dels efectes a llarg termini de l'estrès serien àrees del sistema límbic com l'amígdala i el BST. / Estímulos estresantes básicamente emocionales, como la inmovilización en tabla (IMO), ejercen efectos a largo plazo sobre la actividad del eje hipotalámico-pituitario-adrenal (HPA) en ratas adultas. Estos efectos se caracterizan por una desensibilización, primero a nivel periférico (de adrenocorticotropina (ACTH) y de corticosterona después de unos días) y más tarde a nivel central (de mRNA de corticoliberina (CRF) en el núcleo paraventricular del hipotálamo (PVN) después de unas semanas), de la respuesta de este eje endocrino cuando se expone a los animales al mismo estrés por segunda vez. Este fenómeno presenta similitudes con el síndrome de estrés post-traumático en humanos, y podría enmarcarse dentro del concepto general de plasticidad sináptica. Las preguntas que se pretendieron responder en este trabajo fueron: (i) ¿se pueden generalizar estos efectos a un estrés físico-inmune como la administración de lipopolisacárido bacteriano?, (ii) ¿es el fenómeno específico del estímulo aplicado?, (iii) ¿se correlaciona el fenómeno con la actividad transcripcional temprana en el PVN?, y (iv) ¿cuáles son las áreas cerebrales sensibles a una experiencia previa con estrés? Se utilizaron ratas macho adultas de la cepa Sprague-Dawley, que se estresaron solo en dos ccasiones por IMO o LPS, separadas en el tiempo entre 1 y 4 semanas. Se analizaron en plasma ACTH y corticosterona por radioinmunoanálisis, y factor de necrosis tumoral (TNF)-a mediante ELISA. Se determinaron los niveles de mRNA de c-fos, CRF y receptor de glucocorticoides (GR) y de hnRNA de CRF y vasopresina (AVP) por hibridación in situ. El LPS redujo la respuesta del mRNA de CRF en el PVN al mismo estímulo 4, pero no 2, semanas más tarde; la respuesta periférica y la del TNF-a también fue menor, tanto 1 como 4 semanas más tarde. El LPS bloqueó la hipotermia por LPS 4 semanas más tarde. No se observó desensibilización cruzada entre IMO y LPS en ninguno de los parámetros del eje HPA estudiados. La IMO previa redujo la respuesta del mRNA de c-fos a la IMO en el PVN, tanto 1 como 4 semanas después, y la del hnRNA de CRF a las 4 semanas. Se observaron efectos similares con LPS a nivel del mRNA de c-fos, pero no de hnRNA de CRF, a las 4 semanas. La IMO o el LPS previos no alteraron la respuesta del hnRNA de AVP al mismo estímulo. En general, no se observaron efectos cruzados consistentes entre IMO y LPS a nivel de hnRNA de CRF o de AVP, o de mRNA de c-fos en el PVN. La IMO o el LPS no modificaron los niveles basales de mRNA de GR en la formación hipocampal y en el PVN 4 semanas después. El patrón de activación cerebral del mRNA de c-fos por IMO o LPS fué distinto. Las áreas cerebrales sensibles a la IMO previa fueron la división ventral del septum lateral, el PVN, la amígdala medial, la división medial ventral del núcleo de la estría terminal (BST), el locus coeruleus (LC) y el núcleo del haz solitario. En el caso del LPS, estas áreas fueron el PVN, la amígdala central, la división lateral del BST y el LC. De los resultados obtenidos se desprende que los efectos a largo plazo del estrés se pueden generalizar a estímulos de naturaleza física-inmune como el LPS. Además, el fenómeno parece específico del estímulo aplicado y se detecta también en la actividad transcripcional temprana en el PVN. Finalmente, postulamos que las estructuras cerebrales más probablemente responsables de los efectos a largo plazo del estrés serían áreas del sistema límbico como la amígdala y el BST. / Emotional stressors, such as immobilisation (IMO), can exert long-term effects on the hypothalamic-pituitary-axis (HPA) activity in adult rats. These effects are characterised by a peripheral (adrenocorticotropin (ACTH) and corticosterone, days later) and central (corticotropin-releasing factor (CRF) in the PVN, weeks later) desensitisation of the response of this endocrine axis when the animals are submitted to the same stressor a second time. This phenomenon shares some commonalities with posttraumatic stress disorder in humans, and might fall within the framework of neuroplasticity.The aims of this work attempted to adress the following questions: (i) can this effects be generalised to stressors of physical-immunological nature such as lipopolysaccharide (LPS)?, (ii) is this phenomenon specific for the stressor applied?, (iii) is this phenomenon reflected in the early transcriptional activity in the PVN?, and (iv) which are the brain areas sensitive to a single previous stress exposure?Male Sprague-Dawley adult rats were stressed only two times by IMO or LPS, 1 or 4 weeks ahead. Plasma ACTH and corticosterone were determined by radioinmunoassay, and tumor necorsis factor (TNF)-a by ELISA. C-fos mRNA , CRF and glucocorticoid receptor (GR) and CRF and AVP hnRNA levels were quantifyed by in situ hybridisation.Previous LPS reduced the CRF mRNA response in the PVN to the same stimulus 4, but not 2, weeks later; the peripheral response, and also the response of TNF-a, was also reduced, both 1 and 4 weeks later. Previous LPS abolished the LPS-induced hypothermia 4 weeks later. There was no cross-desensitisation between IMO and LPS in any of the HPA axis parameters studied. Previous IMO reduced the c-fos mRNA response to IMO in the PVN, both 1 and 4 weeks later, and also the CRF hnRNA response only at 4 weeks. Similar effects were observed with LPS on c-fos mRNA, but not CRF hnRNA, at 4 weeks. Previous IMO or LPS did not modify the AVP hnRNA response to the same stimulus. In general, there were no consitent cross-desensitisation effects between IMO and LPS on CRF or AVO hnRNA, or c-fos mRNA in the PVN. IMO or LPS did not modify basal GR mRNA levels in the hippoccampal formation or PVN 4 weeks later.There was a different pattern of c-fos mRNA activation in the brain by IMO or LPS. The areas sensitive to a previous IMO session were the ventral dicision of the lateral septum, PVN, medial amigdala, medial ventral division of the bed nucleus (BST) locus coeruleus (LC) and nucleus of the solitary tract. In the case of LPS, these areas were the PVN, central amigdala, lateral division of the BST and LC.Freom these results we can conclude that long-term effects of stress can be generalised to stressors of physical-immunological nature such as LPS. In addition, this phenomenon seems to be stressor-specific and can be detected on the early transcriptional activity in the PVN. Finally, we suggest that the brain sites that would most probably be responsible of the long-term effects of stress are areas of the lymbic system such as the amigdala and BST.

名古屋大学博士学位論文 学位の種類 : 医学博士(論文) 学位授与年月日:平成4年9月22日 大野元嗣氏の博士論文として提出された

Thesis (Ph. D.)--University of Massachusetts Amherst, 2009. / Open access. Includes bibliographical references (p. 89-87). Print copy also available.

Adrenal gland, as an important effector tissue of the sympathetic nervous system, has critical roles in cardiovascular system under both chronic hypoxia (CH) and intermittent hypoxia (IH) conditions. Nitric oxide (NO), synthesized by nitric oxide synthases (NOS), is the most important intracellular signaling molecule, as well as free radical in response to hypoxia. Yet the regulation and effects of endogenous NO production mediated in the adrenal medulla induced by hypoxia remains largely unknown. We first studied how endogenous NO production were regulated by different NOS in rat adrenal medulla in response to CH or IH. After CH, elevated levels of endogenous NO production, eNOS expression, and apoptotic chromaffin cells were observed in the adrenal medulla. However, a remarkable decreased endogenous NO production and nNOS expression were shown in the IH-treated adrenal medulla. These results suggested that, in the rat adrenal medulla, the elevation of NO production through increased protein level of eNOS may play a protective role in the adaptive response to CH; the reduction of NO production through decreased expression of nNOS is important for the pathophysiological response to IH. The oxidative stress and cellular injury in the adrenal medulla under chronic intermittent hypoxia (CIH) condition is undefined. We tested the hypothesis that melatonin, a potent antioxidant, is protective against CIH-induced oxidative stress and local inflammation in the rat adrenal medulla. Results showed that levels of oxidative stress, lipid peroxidation, and inflammatory mediators were significantly increased after CIH treatment. Also, the protein levels of antioxidant enzymes were significantly lowered in the hypoxic group. Co-treatment of melatonin with hypoxia significantly reduced oxidative stress and inflammatory responses in the adrenal medulla. Moreover, the amount of apoptotic cells in the hypoxic groups was significantly less in the melatonin-treated group. Thus, melatonin may act as a protective agent against adrenal damages in patients with severe obstructive sleep apnea syndrome. Previous studies have shown that CIH associated with recurrent apnea induced oxidative stress and pathophysiological changes in the cardiovascular system. Yet the mechanism of the CIH-induced oxidative stress and local inflammation in the adrenal medulla was undefined. We therefore determined whether the up-regulation of the expression of NADPH oxidase (NOX) mediated by renin-angiotensin system (RAS) may take part in the injuries caused by CIH in the rat adrenal medulla. We found that CIH treatment dramatically induced marker levels of adrenal oxidative stress, inflammation, macrophage infiltration, and apoptosis in rats. Co-treatment with NOX inhibitor, apocynin, counteracted such reactions. Furthermore, the mRNA levels of NOX subunits (p22PHOX, NOX2, and NOX4) and RAS components (ATG, AT1, and AT2) were increased significantly in the CIH group, but reduced in apocynin-treated CIH group, supporting the involvement of NOX and RAS in CIH-induced adrenal injury. In conclusion, we defined the roles of NO production, NOX, and RAS in the rat adrenal injury during hypoxic conditions. We also found that melatonin can protect adrenal medulla from hypoxia-induced damages in rat. / published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

Multiple endocrine neoplasia syndrome type 1 (MEN1) is an autosomal dominantly inherited disease, which is described as an association of tumors mainly in endocrine organs, including pancreas and adrenal glands. Pancreatic neuroendocrine tumors (PNETs) are the most common cause of death in MEN1 patients. More than one third of the MEN1 patients also develop enlargement of the adrenals. MEN1 is caused by a germline mutation of MEN1 gene, a tumor suppressor gene that is located on the human chromosome 11. As noticed, the MEN1 related tumors often develop prior to inactivation of both wild type alleles, indicating MEN1 haploinsufficiency. In this thesis, I utilized a conventional Men1 mouse model that has the phenotype mimicking the human MEN 1 traits, in order to investigate MEN1 tumorigenesis in endocrine pancreas and adrenal glands.   The microvascular aberrations contributing to development and maintenance of PNETs were characterized. The increased vascular density of PNETs developed in the Men1 mice was paralleled by an early and extensive redistribution of pericytes within endocrine tissue. These morphological alterations were supported by fine-tuned variations in expression of several angiogenic regulators  (VEGF, FGF and PDGF) and were further potentiated by hypoxia. Vascular reactivity and blood perfusion of tumor arterioles were significantly altered in response to glucose and L-nitro-arginine methyl ester. Investigation of adrenals from10-month-old Men1 mice showed 681 proteins in mass spectrometry data sets, in which 52 proteins were commonly found in the Men1+/+ and Men1+/- adrenals, and the differential expression between the genotypes reached significant levels. Prdx3, catalyzing the reduction of oxidative stress to cell survival, is one of the overexpressed proteins. Some proteins belonging to the PPARα pathway, e.g. ACLY were also overexpressed. Subsequent microRNA (miRNA) profiling analysis of adrenals from the same age group revealed 31 miRNAs whose expression was significantly altered in comparison between the genotypes. The tumor suppressor miRNAs, miR-486, miR-330 and miR-214, were significantly downregulated in Men1+/- adrenals. The latter, miR-214, is known to inhibit ACLY expression. This finding was in concordance with the proteomic analysis. The oncogene miRNAs, miR-132 and miR-494, were significantly enhanced in the Men1+/- adrenals. Gene ontology analysis demonstrated overrepresentation of the miRNA-targeted genes that are involved in nucleic acid metabolism, vasculature development, angiogenesis, and transcription. Together, these finding after validation in humans may be exploited to improve MEN1 cancer treatment.