京都大学 / 新制・論文博士 / 博士(医学) / 乙第13452号 / 論医博第2245号 / 新制||医||1054(附属図書館) / (主査)教授 椛島 健治, 教授 森信 暁雄, 教授 浅野 雅秀 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

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As urbanization spreads, understanding its impact on wildlife is increasingly urgent. By comparing the traits and fitness of individuals within the same species found in both urban and rural habitats (urban adapters), we can better understand the behavioral and physiological coping mechanisms wild birds employ in the face of rapid environmental change. For my dissertation, I investigated the physiological, behavioral, and fitness differences between urban and rural living song sparrows (Melospiza melodia) to explore how song sparrows are adjusting to urban environments. In my first chapter, I investigated urban birds' termination of the glucocorticoid stress response by looking at their ability to reduce circulating levels of glucocorticoid 'stress' hormones and the relative abundance of receptors that provide negative feedback in the hippocampus and hypothalamus. I found that urban males have a lower relative abundance of glucocorticoid receptors and the enzyme 11β-HSD2 in the hippocampus compered too rural, though we found no difference in negative feedback at the periphery, as both urban and rural song sparrows responded similarly to a challenge with synthetic glucocorticoid (dexamethasone). In chapter 2, I asked if increased aggression, which has been rigorously documented in urban males, is also expressed by females, and whether this aggressive signaling is constraining other reproductive behaviors such as maternal care. Indeed, female song sparrows, like males, expressed increased aggressive signaling compared to rural, suggesting urban habitats may favor a more aggressive phenotype. Finally, in Chapter 3, I investigated the consequences of increased male aggression on their social partners and offspring by measuring parental care and nestling outcomes across urban and rural habits. I was unable to establish a trade-off between parental care and aggression in either sex, suggesting this increased aggression is not constraining other reproductive behaviors. In fact, the more aggressive urban males visited the nest significantly more frequently, a trend also seen in urban females during the daylight hours, though the relationship was not significant over a 24-hour period. Additionally, urban birds had significantly higher reproductive metrics compared to rural, though they also had the added cost of increase brood parasitism by brown-headed cowbirds (Molothrus ater) compared to rural. Overall increased urban aggression was associated with higher reproductive success without any reduction in paternal care. Additionally, we found physiological differences in the glucocorticoid stress response system associated with the differences in habitat but whether theses differences represent mechanisms of acclimation or potential costs of living in urban habitats is not yet clear. / Doctor of Philosophy / As urbanization spreads, understanding its impacts on wild bird conservation is increasingly urgent. By comparing the behaviors and reproductive success of animals living in urban and rural habitats (urban adapters), we can better understand the coping mechanisms wild birds' employ in the face of this form of rapid environmental change. In my dissertation, I compared the physiology, behavior, and reproductive success of urban and rural song sparrows (Melospiza melodia) to explore the changes song sparrows make to survive and reproduce in urban environments. In my first chapter, I investigated how urban birds terminate their stress response by looking at their ability to reduce circulating levels of stress hormones and the relative abundance of "shut down" targets in the brain. In chapter 2, I asked if increased aggression, regularly document in urban males, is also expressed by females, and whether this aggression is constraining other reproductive behaviors. Finally, in Chapter 3, I investigated the consequences of increased male aggression on their social partners and offspring by measuring parental care and nestling outcomes across urban and rural habits. I found that urban males have a lower relative abundance of one type of "shut down" target, and a lower abundance of a potentially protective enzyme in the hippocampus, though we found no difference in how quickly urban and rural birds cleared stress hormone from their blood. Female song sparrows, like males, expressed increased aggressive signaling compared to rural, suggesting urban habitats may favor a more aggressive pattern of behavior. However, I was unable to establish a trade-off between parental care and aggression in either sex, suggesting increased aggression is not constraining other reproductive behaviors. In fact, the more aggressive urban males visited the nest significantly more often, a trend also seen in urban females during the daylight hours, though the relationship was not significant over a 24-hour period. Additionally, urban birds had significantly higher reproductive metrics compared to rural, though they also had the added energetic cost of increased brood parasitism by brown-headed cowbirds (Molothrus ater), compared to rural. Collectively, my results suggest that individuals of this species, the song sparrow, may benefit from livening in low intensity urban habitats and that living in such altered environments favors or permits higher aggression.

Many aspects of biological function are affected by hormones, from physiology to behavior, and the synthesis and release of hormones in vertebrates are regulated by the endocrine axes of control. A growing body of research shows that the mechanisms underlying the endocrine axes of control are complex and interconnected, with many hormones having multiple effects, and with many interactions between axes. In this study, I examined the effects of decreased thyroid function on the glucocorticoid stress response in Japanese quail, a potential interaction between the hypothalamic-pituitary thyroid (HPT) and hypothalamic-pituitary adrenal (HPA) axes of control. I used the thyroid inhibitor ammonium perchlorate (AP) for 2 weeks and 5 weeks to induce two states of decreased thyroid function: a thyroid challenged state, in which birds have depleted thyroidal T4 content, but still maintain euthyroid (normal) concentrations of plasma T4, and a hypothyroid state, in which birds have depleted thyroidal T4 content and decreased concentrations of plasma T4. Thyroid function was assessed by measuring plasma T4 concentrations, thyroidal T4 content, and thyroid gland mass. I took blood samples from birds both immediately prior to and immediately following a 30 minute confinement and agitation stressor to evaluate the effects of decreases in thyroid function on basal and stress-induced plasma corticosterone and plasma T4 concentrations. I found two key results: First, although baseline levels of plasma corticosterone were unchanged, the corticosterone stress response was significantly blunted in both the thyroid challenged and hypothyroid birds as compared to controls. This finding suggests that the HPT and HPA axes are functionally connected in birds, and other evidence suggests this connection is likely at the pituitary or hypothalamic level. Second, in hypothyroid birds, plasma T4 concentrations were elevated (into the euthyroid range) in response to the experimental stressor, although no change in plasma T4 was observed in thyroid challenged or control birds. This finding suggests that plasma T4 may have a permissive role in mounting a stress response. / Master of Science in Life Sciences

Parasitic infections are ubiquitous, but the consequences to hosts can vary substantially. Variation in the consequences of infection can be related to individual differences in the use of two parasite defense strategies, resistance and tolerance. Resistance entails reducing parasite burden by removing parasites or restricting parasite reproduction. Tolerance involves minimizing the costs associated with a given parasite burden. Genetic variation, environmental conditions, and life history stage can contribute to variation in resistance and tolerance, but the physiological mechanisms that underlie investment in each strategy are not well understood. I proposed that glucocorticoid hormones, which mediate responses to challenges in the physical and social environment in vertebrates, might alter host investment in resistance and tolerance (Chapter I). Glucocorticoids influence a suite of physiological processes including immune function, resource allocation, and tissue growth, all which could alter resistance and tolerance. Using a combination of observational and experimental studies, I test the hypothesis that glucocorticoids mediate resistance and tolerance to infection in red-winged blackbirds (Agelaius phoeniceus) infected with Haemosporidians, including malaria (Plasmodium) and malaria-like (Haemoproteus and Leucocytozoon) parasites. I performed a medication experiment (Chapter II) to identify the physiological consequences of Haemosporidian infection and explored the relationships between glucocorticoids and parasite resistance and tolerance in both an observational field study and a hormone manipulation experiment (Chapters III and IV). Medication treatment effectively reduced Plasmodium burden, increased hematocrit and hemoglobin, and reduced the rate of red blood cell production (Chapter II). In an observational field study (Chapter III), red-winged blackbirds with higher plasma glucocorticoid concentrations maintained higher hematocrit than expected for their parasite burdens, suggesting a positive association between glucocorticoids and tolerance. In this study, I found no support for a relationship between glucocorticoids and resistance. However, experimental elevation of glucocorticoids (Chapter IV) yielded nearly opposite results: the higher of two doses of glucocorticoids increased Plasmodium burdens and caused a decrease in body mass with increasing parasite burden, indicative of a decrease in tolerance. I discuss possible causes of the differences in our observational and experimental studies and the implications of my work for future studies of individual variation in parasite tolerance (Chapter V). / Ph. D.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

Thesis (Ph. D.)--Medical College of Ohio, 2003. / "In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Medical Sciences." Major advisor: Edwin Sanchez. Includes abstract. Document formatted into pages: iv, 126 p. Title from title page of PDF document. Includes bibliographical references (p. 100-124).

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: The synthetic progestins, medroxyprogesterone acetate (MPA) and norethisterone (Net) and its derivatives (norethisterone enanthate (Net-EN) and norethisterone acetate (Net-A)), are widely used as contraceptives and in hormone replacement therapy (HRT). Several studies have indicated that synthetic progestins modulate immune function and increase the risk of sexually transmitted infections. However, little is known about the molecular mechanism of action of MPA and Net, in particular their regulation of gene expression in the female genital tract, as compared to progesterone (P4). In the first part of this thesis, the effect of P4, MPA and Net-A on the expression of the endogenous chemokine genes, macrophage inflammatory protein (MIP)-1α and MIP-1β, was investigated in a human vaginal epithelial cell line (Vk2/E6E7). Quantitative realtime PCR (QPCR) showed that both P4 and MPA upregulated the TNF-α-induced expression of MIP-1α and MIP-1β mRNA, while Net-A had no effect. Using siRNA technology, it was found that the responses to P4 and MPA on the MIP-1α gene, but not the MIP-1β gene, are mediated via the glucocorticoid receptor (GR). In the second part of the thesis, it was investigated whether the HIV-1 accessory protein, viral protein R (Vpr), could modulate the action of ligands on MIP-1α and MIP-1β gene expression. QPCR showed that Vpr abrogates the effects of P4 and MPA on the TNF-α induced expression of MIP-1α and MIP-1β. Silencing the GR with siRNA technology showed that the GR plays a role in the effect of Vpr on the P4 and MPA-induced expression of MIP-1α. Taken together, these results show that MPA and Net-A display differential effects on chemokine gene expression in a human vaginal epithelial cell line. Furthermore, this study shows that Vpr modulates the effects of MPA bound to the GR. Thus, the results of this thesis provide insight into the effect of synthetic progestins on the immune response in the vagina, and possibly how HIV-infection may alter these responses. / AFRIKAANSE OPSOMMING: Die sintetiese progestiene medroksieprogesteroon asetaat (MPA) en noretisteroon (Net) en derivate daarvan (noretisteroon enantaat (Net-EN) en noretisteroon asetaat (Net-A)), word op grootskaal gebruik as voorbehoedmiddels en in hormoonvervangingsterapie (HVT). Verskeie studies het al aangedui dat sintetiese progestiene immuunfunksie moduleer en die risiko vir seksuel oordraagbare infeksies verhoog. Daar is egter min bekend oor die molekulêre meganisme van aksie van MPA en Net, in die besonder die regulering van geenuitdrukking in die vroulike geslagskanaal in vergelyking met progesteroon (P4). In die eerste deel van hierdie tesis is die effek van P4, MPA en Net-A op die uitdrukking van endogene chemokiene gene, makrofaag inflammatoriese proteïen (MIP)-1α en MIP-1β, in 'n menslike vaginale epiteel sellyn (Vk2/E6E7) bestudeer. Kwantitatiewe intydse PKR (KPKR) het getoon dat beide P4 en MPA die TNF-α-geïnduseerde uitdrukking van beide die MIP-1α en MIP-1β mRNA uitdrukking op reguleer, terwyl Net-A geen effek getoon het nie. Met die gebruik van siRNA-tegnologie is daar bevind dat die effekte van P4 en MPA, bemiddel word deur die glukokortikoïd-reseptor (GR) op MIP-1α geen uitdrukking, maar nie op MIP-1β nie. In die tweede deel van die tesis, is ondersoek of die MIV-1-bykomstigheidsproteïen, virale proteïen R (Vpr), die aksie van die ligande op MIP 1α en MIP-1β geenuitdrukking kan moduleer. KPKR toon dat Vpr die uitwerking van P4 en MPA op die TNF-α-geïnduseerde uitdrukking van MIP 1α en MIP-1β kanselleer. Die verwydering van die GR met siRNA-tegnologie toon dat die GR 'n rol in die uitwerking van Vpr op die P4 en MPA-geïnduseerde uitdrukking van MIP-1α speel. Ter samevatting: hierdie resultate toon dat MPA en Net-A differensiële uitwerkings vertoon op chemokiene geenuitdrukking in 'n menslike vaginale epiteel sellyn, en dat Vpr hierdie uitwerkings moduleer van MPA gobonde aan die GR. Die resultate van hierdie tesis werp dus lig tot die uitwerking van sintetiese progestiene op die immuunreaksie in die vagina, sowel as hoe MIVinfeksie hierdie reaksies kan verander.

During stress conditions, glucocorticoids are secreted and exert most of their physiological responses by binding to and modulating the transcriptional activity of the glucocorticoid receptor (GR). Once activated, GR can regulate numerous cellular processes including inflammation, development, growth, metabolism and apoptosis. Although glucocorticoids have been used in the treatment of leukaemia for over 50 years, with the molecular mechanisms by which steroids exert their pro-apoptotic effect, the pathways responsible for the development of resistance to glucocorticoid treatment, as well as their role in the programmed cell death in other tissue types have not been precisely defined. Research has demonstrated that glucocorticoid-induced apoptosis requires a transcriptionally active form of GR and is executed by the induction of the intrinsic pathway of apoptosis. In addition, GR is regulated by diverse types of cytotoxic stress; including UV irradiation and hypoxia, which alter the receptor’s transcriptional activity through multiple mechanisms. These include post-translational modifications, subcellular localisation and interaction of the receptor with co-regulator proteins. The aims of this study are to identify novel members of the Bcl-2 family that are regulated at the transcriptional level by GR in both leukaemia and other tissue types where glucocorticoids promote cell survival. In addition, the molecular crosstalk between signalling pathways activated by cytotoxic stress conditions and the mechanisms by which they differentially modulate the apoptotic response will be investigated. Results obtained in this study have identified putative glucocorticoid response elements in the promoters of the BH3-only pro-apoptotic gene NOXA and the anti-apoptotic gene Mcl-1 and confirmed that both NOXA and Mcl-1 are direct GR transcriptional targets. The glucocorticoid-mediated expression of NOXA and Mcl-1 alters their protein-protein interaction pattern, leading to the subsequent destabilisation of Mcl-1 in cell lines that undergo glucocorticoid-induced apoptosis. Investigation into the effects that other cytotoxic stress pathways have on GR function have revealed that serine 226 phosphorylation of GR by JNK occurs in a rapid and transient manner. Phosphorylation has inhibitory effects on the transcription of GR targets in a gene-specific manner, including the differential regulation of NOXA gene expression. During hypoxia, glucocorticoids differentially regulate the GR and HIF-1 target genes, NOXA and Mcl-1, altering the apoptotic response. This study has provided additional insight into the molecular mechanisms that govern glucocorticoid-induced programmed cell death and revealed mechanisms by which glucocorticoids and cytotoxic stress pathways crosstalk, regulating apoptosis.

Doxorubicin is one of the most potent anticancer drugs used in the treatment of wide spectrum of neoplastic diseases including breast, thyroid, colon and liver cancer. However, doxorubicin use is associated with undesirable side effects including cardiomyopathy and congestive heart failure. In the present study we have established that treatment of rat cardiac myoblasts (H9c2 cells) with doxorubicin resulted in H9c2 cell injury in a dose and time dependent manner with almost 50% cell death obtained at 5 μM of doxorubicin treatment for 24 hours. We have selected about 50% cell injury as optimum doxorubicin-induced cell injury because once this threshold is reached, cells became irreversibly injured and could not respond to protective treatment. Another potent antineoplastic drug cyclophosphamide had no cardiotoxic effects on H9c2 cells even at 35 μM concentration and up to 72 hours of treatment. Pretreatment of H9c2 cells for 24 hours with dexamethasone, cortisol, corticosterone or progesterone, significantly protect H9c2 myoblasts against subsequent 5 μM doxorubicin treatment for 24 hours in a concentration dependent manner with maximum protection obtained at 100 nM dexamethasone, 100 nM progesterone, 500 nM cortisol and 500 nM corticosterone. However, testosterone or dehydroepiandrosterone had no protective effects even at 10 μM concentration. It is concluded that both glucocorticoids and progesterone protect H9c2 cells against doxorubicin-induced cell injury.