Cadmium (Cd) is a nonessential metal present in sublethal concentrations within the aquatic environment. Cd is an endocrine disruptor and high concentrations of this metal suppress stressor-induced cortisol production in fish. However, few studies have examined the effect of Cd at concentrations that are environmentally relevant on the functioning of the hypothalamus-pituitary-interrenal (HPI) axis. The HPI axis activity is essential in the stressor-induced cortisol production, a highly conserved adaptive response to stress in vertebrates. Elevation of plasma glucose in response to a rise in plasma cortisol is mediated through steroid activation of glucocorticoid receptors (GRs), but the mechanism of action of Cd in disrupting target tissue cortisol action is not known in fish. The overall objective of this thesis was to examine the impact of sublethal and environmentally relevant levels of Cd on the stress response and target tissue metabolic capacities, and to investigate the mechanisms of action of this metal in disrupting cortisol production and target tissue cortisol action in rainbow trout (Oncorhynchus mykiss). The impact of subchronic exposure to environmentally relevant levels of Cd on metabolic capacity and stress performance was identified through a 28 day (d) in vivo exposure of juvenile rainbow trout to either of two Cd concentrations (0.75 µg/L or 2.0 µg/L). During the exposure period, juvenile rainbow trout accumulated Cd within the liver, kidney and gills, but were able to adapt to exposure concentrations as no changes were observed in plasma cortisol, glucose and lactate levels. However, changes in abundance of mRNAs encoding proteins involved in corticosteroidogenesis, including melanocortin 2 receptor (MC2R), steroidogenic acute regulatory protein (StAR) and P450 side chain cleavage enzyme (P450scc), and liver GR protein expression suggesting endocrine disruption over the 28 d period. Also, target tissue metabolic capacities, including lower liver glycogen content and changes in intermediary metabolic enzyme activities in the liver and gill, were compromised by the 28 d exposure to Cd. The response to a secondary handling stressor at either 7 or 28 d exposure was attenuated suggesting that subchronic exposure to low levels of Cd disrupts the highly conserved adaptive stress response in rainbow trout. Upon further investigation using in vitro head kidney slices exposed to 0, 10, 100 or 1000 nM of Cd and stimulated with adrenocorticotropic hormone (ACTH), a similar inhibition of cortisol production was observed, as demonstrated in vivo, suggesting that Cd disrupts interrenal corticosteroidogenesis in fish. The impact of Cd on ACTH-stimulated cortisol production involved the suppression in the abundances of MC2R, StAR and P450scc transcripts. This response was also mimicked when head kidney slices from 7 d Cd exposed fish were incubated ex vivo with ACTH confirming that interrenal tissue is a key target for endocrine disruption by Cd. In both the in vitro and ex vivo incubations of head kidney slices 8-Bromo-cAMP (a cAMP analog) completely abolished the Cd-mediated cortisol inhibition demonstrating for the first time that Cd disruption of corticosteroidogenesis is occurring upstream of cAMP production. Further investigation of Cd-mediated impact on MC2R showed alterations in MC2R mRNA transcripts during in vivo exposure after 7 days and an attenuation of MC2R mRNA levels after Cd-exposed fish were subjected to a handling stressor. Disruptions in the mRNA abundance of MC2R was associated with disruptions of melanocortin receptor accessory protein 1 (MRAP1), but not MRAP2; a phenomenon that was also observed in ex vivo head kidney slices. Cell transfection studies confirmed that rainbow trout MC2R/MRAP1 receptor complex displayed decreased activity in the presence of Cd. Taken together these results suggest that Cd directly targets the MC2R/MRAP1 complex to inhibit ACTH-stimulated cortisol production in juvenile rainbow trout. In addition to Cd inhibiting interrenal steroidogenesis, the results also suggest that Cd may impact the negative feedback regulation of cortisol through the suppression of brain mineralocorticoid receptor (MR), but this requires further investigation. At the target tissue level, Cd by itself did not significantly affect liver metabolism, but inhibited the cortisol-induced glucose production in liver slices. This involved suppression of GR protein expression along with the suppression of GR-responsive genes, including phosphoenolpyruvate carboxykinase (PEPCK) and suppressor of cytokines signaling 1 (SOCS1) and changes in enzyme activities, including hexokinase, glucokinase, pyruvate kinase and PEPCK, pointing to a disruption in liver GR signaling by Cd. Altogether, Cd exposure disrupts the organismal stress responses in juvenile rainbow trout. Furthermore, Cd impairs the ability of juvenile rainbow trout to respond to a secondary stressor, which is a vital adaptive process that is fundamental to successful stress performance. Most importantly, these studies highlight for the first time that disruption of the HPI axis to attenuate cortisol production occurs at the level of the MC2R/MRAP1 complex, suggesting that the mechanism of action for attenuation of cortisol occurs at the level of MC2R activation. Also, GR signaling is a key target for Cd and may be a mechanism leading to altered metabolic capacities in stressed fish from Cd-contaminated sites. Overall environmentally relevant levels of Cd disrupt cortisol production and target tissue action of this steroid in rainbow trout.

Cadmium (Cd) is a nonessential metal present in sublethal concentrations within the aquatic environment. Cd is an endocrine disruptor and high concentrations of this metal suppress stressor-induced cortisol production in fish. However, few studies have examined the effect of Cd at concentrations that are environmentally relevant on the functioning of the hypothalamus-pituitary-interrenal (HPI) axis. The HPI axis activity is essential in the stressor-induced cortisol production, a highly conserved adaptive response to stress in vertebrates. Elevation of plasma glucose in response to a rise in plasma cortisol is mediated through steroid activation of glucocorticoid receptors (GRs), but the mechanism of action of Cd in disrupting target tissue cortisol action is not known in fish. The overall objective of this thesis was to examine the impact of sublethal and environmentally relevant levels of Cd on the stress response and target tissue metabolic capacities, and to investigate the mechanisms of action of this metal in disrupting cortisol production and target tissue cortisol action in rainbow trout (Oncorhynchus mykiss). The impact of subchronic exposure to environmentally relevant levels of Cd on metabolic capacity and stress performance was identified through a 28 day (d) in vivo exposure of juvenile rainbow trout to either of two Cd concentrations (0.75 µg/L or 2.0 µg/L). During the exposure period, juvenile rainbow trout accumulated Cd within the liver, kidney and gills, but were able to adapt to exposure concentrations as no changes were observed in plasma cortisol, glucose and lactate levels. However, changes in abundance of mRNAs encoding proteins involved in corticosteroidogenesis, including melanocortin 2 receptor (MC2R), steroidogenic acute regulatory protein (StAR) and P450 side chain cleavage enzyme (P450scc), and liver GR protein expression suggesting endocrine disruption over the 28 d period. Also, target tissue metabolic capacities, including lower liver glycogen content and changes in intermediary metabolic enzyme activities in the liver and gill, were compromised by the 28 d exposure to Cd. The response to a secondary handling stressor at either 7 or 28 d exposure was attenuated suggesting that subchronic exposure to low levels of Cd disrupts the highly conserved adaptive stress response in rainbow trout. Upon further investigation using in vitro head kidney slices exposed to 0, 10, 100 or 1000 nM of Cd and stimulated with adrenocorticotropic hormone (ACTH), a similar inhibition of cortisol production was observed, as demonstrated in vivo, suggesting that Cd disrupts interrenal corticosteroidogenesis in fish. The impact of Cd on ACTH-stimulated cortisol production involved the suppression in the abundances of MC2R, StAR and P450scc transcripts. This response was also mimicked when head kidney slices from 7 d Cd exposed fish were incubated ex vivo with ACTH confirming that interrenal tissue is a key target for endocrine disruption by Cd. In both the in vitro and ex vivo incubations of head kidney slices 8-Bromo-cAMP (a cAMP analog) completely abolished the Cd-mediated cortisol inhibition demonstrating for the first time that Cd disruption of corticosteroidogenesis is occurring upstream of cAMP production. Further investigation of Cd-mediated impact on MC2R showed alterations in MC2R mRNA transcripts during in vivo exposure after 7 days and an attenuation of MC2R mRNA levels after Cd-exposed fish were subjected to a handling stressor. Disruptions in the mRNA abundance of MC2R was associated with disruptions of melanocortin receptor accessory protein 1 (MRAP1), but not MRAP2; a phenomenon that was also observed in ex vivo head kidney slices. Cell transfection studies confirmed that rainbow trout MC2R/MRAP1 receptor complex displayed decreased activity in the presence of Cd. Taken together these results suggest that Cd directly targets the MC2R/MRAP1 complex to inhibit ACTH-stimulated cortisol production in juvenile rainbow trout. In addition to Cd inhibiting interrenal steroidogenesis, the results also suggest that Cd may impact the negative feedback regulation of cortisol through the suppression of brain mineralocorticoid receptor (MR), but this requires further investigation. At the target tissue level, Cd by itself did not significantly affect liver metabolism, but inhibited the cortisol-induced glucose production in liver slices. This involved suppression of GR protein expression along with the suppression of GR-responsive genes, including phosphoenolpyruvate carboxykinase (PEPCK) and suppressor of cytokines signaling 1 (SOCS1) and changes in enzyme activities, including hexokinase, glucokinase, pyruvate kinase and PEPCK, pointing to a disruption in liver GR signaling by Cd. Altogether, Cd exposure disrupts the organismal stress responses in juvenile rainbow trout. Furthermore, Cd impairs the ability of juvenile rainbow trout to respond to a secondary stressor, which is a vital adaptive process that is fundamental to successful stress performance. Most importantly, these studies highlight for the first time that disruption of the HPI axis to attenuate cortisol production occurs at the level of the MC2R/MRAP1 complex, suggesting that the mechanism of action for attenuation of cortisol occurs at the level of MC2R activation. Also, GR signaling is a key target for Cd and may be a mechanism leading to altered metabolic capacities in stressed fish from Cd-contaminated sites. Overall environmentally relevant levels of Cd disrupt cortisol production and target tissue action of this steroid in rainbow trout.

The turquoise killifish Nothobranchius furzeri is an increasingly popular model species for comparative vertebrate research. As such, the basic physiology, including responses to one or multiple stress events are of primary interest to the present study. After successful rearing, adult male and female killifish were exposed to one or more acute confinement events. Whole-blood was taken from adult males, in addition to male and female whole-body samples for cortisol analysis. Separate adults were also sampled for tissue specific expression of corticotropin releasing hormone (CRH), mineralocorticoid receptor (MR), and glucocorticoid receptor (GR) mRNA within the cranium, gills, and liver. Following a 30 minute confinement stress, male plasma cortisol significantly differed from baseline at 30 minutes (P=0.04). Similarly, both male and female whole-body cortisol were significantly increased (P=0.004 and P=0.04, respectively) at 15 and 30 minutes post-stress. Whole-body cortisol did not differ between the sexes at any sampling point, however; expression of gill MR at 15 minutes was significantly higher in males (P=0.05). Exposure to daily repeated confinement affected the cortisol response in both males and females, resulting in lower baseline and 60 minutes post-stress values in repeatedly-stressed males (P=0.04 and P=0.006, respectively) and lower cortisol at 30 and 120 minutes post-stress in repeatedly-stressed females (P=0.04 and P=0.04). Repeated exposure also resulted in increased cranial CRH and MR at 15 minutes post-stress (P=0.02 and P=0.05, respectively) compared to singly-stressed males. In females, repeated exposure increased gill MR at 120 minutes (P=0.05), but a single stressor resulted in relatively greater expression of cranial CRH at 120 minutes (P=0.02) and MR at 15 minutes (P=0.05). Collectively, the reduced cortisol production observed in repeatedly-stressed adults coupled with only transient changes in receptor expression suggest acclimation to daily stressors can be detected in as little as one week in adult turquoise killifish. This is also the first description of the stress response on this important model species.

Although empirical evidence suggests that interactions occur between serotonin (5- HT) and the hypothalamic-pituitary-interrenal (HPI) axis teleost fish, the mechanisms of serotonergic regulation of the HPI axis require elucidation. The hypothesis that 5-HT regulates the stress response in rainbow trout (Oncorhynchus mykiss) by acting at multiple levels in the HPI axis through tissue-specific 5-HT receptor subtypes was examined. Messenger RNA for 5-HT1A, 5-HT2 and 5-HT4 receptor subtypes was expressed in all HPI axis tissues. Administration of 5-HT in vivo in cannulated trout caused significant increases in plasma cortisol and glucose concentrations. In vitro head kidney preparations revealed a stimulatory effect of 5- HT, acting through the 5-HT4 receptor, on cortisol production. Collectively, these data suggest that 5-HT plays a role in HPI axis activation in rainbow trout, and that at the head kidney level, these effects likely are mediated by the 5-HT4 receptor.

The purpose of this study was to characterize the endocrine stress response of zebrafish (Danio rerio) and to test if this response is modified by sex or the major sex steroids 17β-estradiol (E2) and 11-ketotestosterone (11KT). A standardized stress of having zebrafish swim in a beaker of stirring water resulted in rapid and transient changes in whole-body cortisol. While the cortisol response of males and females were comparable, exposure to E2 (100 ng/L) for 48 h inhibited the cortisol response of males and exposure to 11KT (100ng/L) for 48 h stimulated the cortisol response of females. The inhibitory actions of E2 were mediated in part by decreases in corticotropin releasing factor (crf) expression and decreases in the synthesis of cortisol by interrenal tissue. This research adds to the increasing knowledge of zebrafish physiology and will be useful for future studies concerning the endocrine stress response in fish.

Made available in DSpace on 2014-06-12T18:02:52Z (GMT). No. of bitstreams: 2 arquivo897_1.pdf: 8640800 bytes, checksum: a7ee3c6274f9d3c9d3a9f5fa7142d92d (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2010 / A evolução dos mecanismos de virulência bacteriana está diretamente relacionada com a aquisição dos elementos transponíveis. A compreensão desses fenômenos teve grande avanço com a produção de sequências completas de genomas. Atualmente, sete sequências completas de cepas de Yersinia pestis são conhecidas. Nossas análises dessas sequências revelaram novos aspectos sobre a evolução da ilha de alta patogenicidade (HPI), onde estão as sequências que codificam o sideróforo yersiniabactina (Ybt). As evidências de ciclos adaptativos, mesmo na ausência de genomas intermediários, proporcionaram informações de que a HPI das yersínias foi herdada monofileticamente, podendo a Vibrionaceae ser sua família ancestral. A metodologia foi baseada nas análises das sequências vizinhas às HPIs, assinaturas seletivas dos genes do operon ybt e seus homólogos e frequências do uso de códons dos genomas hospedeiros, que revelaram novos aspectos da evolução desta ilha genômica. Os padrões e as diferenças de conteúdos de GC e das substituições sinônimas e não sinônimas indicam que os genes ybt e seus genomas hospedeiros passaram por diferentes pressões seletivas. Assim, grupos ancestrais diferentes encontraram formas distintas de preservar suas HPIs. Observamos que as sequências vizinhas das HPIs são reguladas por quorum sensing, indicando uma rede geral de regulação que envolve os genes ybt. A análise da organização cromossômica das sete cepas representativas de diferentes regiões do mundo revelou também uma dinâmica de rearranjos que poderia justificar o reconhecimento de novas variantes de Y. pestis até recentemente considerada uma espécie muito homogênea

Production of the glucocorticoid hormone cortisol in response to a stressor is initiated by activation of the hypothalamic-pituitary-interrenal (HPI) axis in fish. Serotonin (5-HT) and negative feedback regulate cortisol production at the whole-animal level; the objective of the present thesis was to investigate their roles in regulating cortisol production by interrenal cells of rainbow trout (Oncorhynchus mykiss). Messenger ribonucleic acid (mRNA) for the 5-HT4 receptor was present in low abundance in interrenal cells. In addition, cortisol production was significantly increased for in vitro head kidney preparations incubated with 5-HT, and this elevated cortisol production was blocked by the 5-HT4 receptor antagonist 5-fluoro-2-methoxy-[1-[2-[(methylsulphonyl) amino] ethyl]-4-piperidinyl]-1h-indole-3-methylcarboxylate sulphamate (GR125487). Thus, 5-HT acts at the head kidney level to regulate cortisol production, probably via the 5-HT4 receptor. Chronic social stress did not appear to regulate the expression of key proteins involved in cortisol biosynthesis or corticosteroid receptors (CR). However, head kidney tissue incubated in vitro with cortisol for 2-8 h showed a reduction in adrenocorticotropic hormone-stimulated cortisol production compared to controls, suggesting the existence of an ultra-short-loop negative feedback mechanism. Thus, the high circulating levels of cortisol in trout experiencing chronic social stress may activate this ultra-short-loop negative feedback mechanism to suppress cortisol production at the head kidney level.

Here I describe the construction and characterization of a new generation of human artificial chromosome that contains an array of DNA sequences that can be used to manipulate the chromosome in vivo and possibly in vitro. This HAC was originated in human fibrosarcoma HT1080 cells from a synthetic alphoid DNA containing an array of TetOperator sequences, cloned in a BAC-based vector. This synthetic ά-satellite DNA formed HACs that were stably maintained throughtout replication and segregation in HT1080 cells. However, I succeeded to also transfer and manipulate the alphoidtetO HAC into a HeLa-based hybrid cell line. The synthetic alphoidtetO HAC chromatin was similar to the chromatin at endogenous centromeric alphoid DNA. Importantly, the DNA sequences embedded in the synthetic HAC were accessible to targeting TetR-fused constructs in vivo. The alphoidtetO HAC could be successfully targeted with a number of TetR:fusion proteins without affecting its chromatin structure, kinetochore assembly and mitotic behaviour. However, the targeting of a transcriptional activator (tTA) inactivated the HAC synthetic alphoidtetO DNA in a fraction of transfected cells. Surprisingly, the targeting of the transcriptional repressor tTS, co-repressor KAP1 or the heterochromatin-associated protein HPIά severely inactivated the synthetic alphoidtetO kinetochore . In fact, upon targeting several inner and outer kinetochore proteins were delocalized from the alphoidtetO sequences. The dissociation of kinetochore proteins CENP-H and CENP-C appeared to precede that of CENP-A. The alphoidtetO HAC lacking inner kinetochore protein complexes showed mitotic defects including misalignment at the metaphase plate and defective anaphase segregation, ultimately being included in tiny DAPI-positive nano-nuclei in the cytoplasm. The transcriptional repressor tTS repressed the low levels of transcription from the alphoidtetO sequences. In addition, targeting of transcriptional repressors altered the HAC chromatin towards a more “closed”, heterochromatic conformation, as seen from the changes in histone tail modifications. Interestingly, the targeting of the histone methyltransferase EZH2 to the alphoidteto HAC showed a much milder inactivating activity compared to KAP1. Based on these results, I propose that the formation of HPI-type of heterochromatin or accumulation of HPIά to the centromeric regions could disrupt the association of constitutive kinetochore proteins to the underlying sequences. Centromeric alphoid sequences lacking a functional kinetochore structure then also loose the centromere-specific histone H3 variant CENP-A becoming definitively inactive. Alternatively, a basal transcriptional activity from centromeric sequences might be required for centromere functionality.

The accumulation of persistent contaminants is a significant issue for the health of aquatic environments. This study aims to determine the effects of polycyclic aromatic hydrocarbons (PAH) on the stress response of fish by monitoring plasma cortisol levels and the expression of key hypothalamic-pituitary-interrenal (HPI) stress axis regulators. Injection of benzo(a)pyrene (BaP), a ubiquitous PAH, induced a differential dose- and time-dependentcortisol response in rainbow trout and brown bullhead. BaP exposure also elicited a species-specific transcriptional response at all levelsof the HPI axis.Similarly, the HPI axis response to a standardized emersionstressor revealed species-specific differences. In the field, exposure of different brown bullhead populations to sediment with complex PAH mixtures did not consistently affect cortisol levels and providedno evidence of genetic adaptation of the stress response. Thus, future studies are needed to bridge the gap in our understanding between the laboratory and field effects of PAHs on the stress response of fish.

In this thesis I present a community of musicians dealing with a specific approach to music of older styles - historically informed performances (HIP). The aim is to answer the question as to why these promising classically educated musicians decided to leave the traditional career path in their field and chose alternative musical expression. I follow the rise and development of the professional scene of historically informed performances and its community from the turn of the 80s and 90s of the 20th century to the present day. Based on the analysis and interpretation of oral historical interviews, I describe changes in this environment and subsequent reflection on the perception of the profession and lives of the actors. I focus on their individual perceptions of authenticity and historical time, formed by their incorporation in early music.