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One unresolved issue in the study of cooperative breeding is why individuals that delay their own reproduction assist others in raising young. Red-cockaded woodpecker helpers may again future indirect fitness benefits by increasing survival of breeders, who produce offspring related to the helper in the future. Breeder survival may be enhanced because of general benefits of group living, either because of the helper's own presence or because helping increases the production of fledglings. I demonstrate that breeder survival increases in the presence of helpers and additional young. Helpers may also reduce the workload of the breeder, and this may increase breeder survival as well. I show that breeders spend less time incubating and provisioning young when a helper is present. Helpers may also gain fitness benefits if once they become breeders, they are assisted by young they previously helped raise. Expected frequencies of reciprocal exchange of helping are low (2%). Slightly higher observed frequencies may be accounted for by preferential helping of kin and effects of territory quality. Reciprocity occurs no more often than expected among helpers unrelated to the young they help raise suggesting that young males do not preferentially helper former care-givers. i conclude that helpers do not gain fitness benefits from reciprocity. I examined the proximate causes of delayed breeding and helping behavior by measuring plasma testosterone (T) and prolactin (PRL) concentrations in female breeders, male breeders, and male helpers during different stages of the reproductive cycle. Among male breeders and helpers, T is low during the nonbreeding stage, peaks during copulation and declines during the incubation and nestling-provisioning stages. Helpers appear physiologically capable of reproducing: their T concentrations are equal those of breeders. Helpers unrelated to the breeding female have higher T than helper related to her. Sexual inactivity by male helpers is best explained by behavioral suppression. Female breeder, male breeder, and male helper PRL was equal and increased from the nonbreeding stage through the copulation and incubation stages. During the nestling provisioning stage, male breeder and male helper PRL declined, while female PRL continued to increase. I conclude that the physiological bases of helping behavior and parental behavior are the same. / Ph. D.

Poor reproductive performance by turkey hens compared with chickens is partially due to the early cessation of egg production associated with the onset of incubation behavior. Prolactin (Prl) is involved in the induction and maintenance of incubation behavior in birds, and understanding the regulatory mechanism(s) of Prl gene expression will provide fundamental information to manipulate Prl production for better reproductive performance in turkey hens. / Ph. D.

No / Sexual dysfunction is a major, although poorly understood, side-effect of treatment with antipsychotic drugs. We have recently show marked disruption of reproductive function and weight gain in female rats treated subchronically with risperidone and haloperidol. The aim of the present study was to examine further the potential relationship between reproductive dysfunction and weight gain in female rats treated with olanzapine. The effects of olanzapine on weight gain, food and water intake, intra-abdominal fat, the oestrous cycle and uterine weight were assessed in group-housed adult female hooded-Lister rats. Olanzapine (0.5-4.0 mg/kg i.p.) or vehicle was administered once daily for 21 days and body weight, food and water intake measured, with histological examination of vaginal lavage to determine the stage of the oestrous cycle. On day 22, animals were sacrificed and intra-abdominal fat, wet and dry uterine weights measured. Olanzapine induced significant weight gain with concomitant increases in food and water intake and intra-abdominal fat without an effect on the oestrous cycle, wet and dry uterine weights or plasma prolactin levels. These results confirm the ability of olanzapine to induce weight gain in female rats on unrestricted normal diet with a concomitant increase in food and water intake and increased intra-abdominal fat. These effects of olanzapine were produced in the absence of any apparent impairment in reproductive function, in contrast to the substantial disruption of oestrous and uterine atrophy previously shown in rats treated with risperidone and haloperidol.

The duplication of genes followed by selection is perhaps the most prominent way in which molecular biological systems gain multiplicity, diversity and functional complexity in evolution. Whole genome duplications (WGDs) therefore have the potential of generating an extraordinary amount of evolutionary innovation. It is now accepted that the vertebrate lineage has gone through two rounds of WGD in its early stages, after the divergence of invertebrate chordates and before the emergence of jawed vertebrates. These basal vertebrate WGDs are called 2R for two rounds of whole genome duplication. An additional WGD called 3R occurred early in the evolution of teleost fishes, before the radiation of this species-rich group. This thesis describes the evolution of several endocrine and neuronal gene families in relation to the vertebrate WGDs, through a comparative genomic approach including both phylogenetic analyses and chromosomal location data across a wide range of vertebrate taxa. These results show that numerous endocrine gene families have expanded in 2R and in several cases also in 3R. These include the gene families of oxytocin and vasopressin receptors (OT/VP-R), somatostatin receptors (SSTR) and insulin-like growth factor binding proteins (IGFBP). For the OT/VP-R and SSTR families, previously undescribed subtypes were identified. The protein hormone family that includes growth hormone (GH), prolactin (PRL) and somatolactin (SL) acquired a new PRL gene in 2R, however the origins of GH, PRL and SL likely predate 2R. The corresponding family of receptors diversified during different time periods through a combination of local duplications and 3R. Neuronal gene families of the visual system have also expanded in 2R and 3R. The results presented here demonstrate that the vertebrate repertoire of visual opsin genes arose in 2R as part of chromosomal blocks that also include the OT/VP-R genes. The gene families including the transducin alpha, beta and gamma subunits also arose in 2R, hinting at the importance of these events in the diversification and specialization of phototransduction cascades for rods and cones. Thus, the whole genome duplications have been important contributors to the evolution of both vision and endocrine regulation in the vertebrates.

A prolactina humana (hPRL) é um hormônio sintetizado pela hipófise com inúmeras funções tais como: lactação, reprodução e regulação osmótica. Este hormônio é frequentemente dosado em casos de problemas na lactação, infertilidade, além de estudos que elucidam sua ligação em alguns tipos de câncer (mama, próstata e útero). A hPRL é encontrada na forma não glicosilada (NG-hPRL) (23 kDa) e glicosilada (G-hPRL) (25 kDa), sendo a isoforma glicosilada um modelo ideal de análise de perfil de N-glicanos, já que possui um único sítio de glicosilação localizado na Asparagina 31. A glicosilação está relacionada diretamente à solubilidade, à estabilidade, ao enovelamento, à meia-vida e atividade biológica in vivo. As células de ovário de hamster chinês (CHO) e as células embrionárias de rim humano (HEK293) são os hospedeiros mais utilizados para expressão de proteínas recombinantes, já que podem ser cultivadas em altas densidades e por possuírem similaridade nas modificações pós-traducionais. O objetivo foi expressar, purificar e realizar uma caracterização físico-química e biológica da hPRL glicosilada de células HEK293, incluindo análise da estrutura de carboidratos. Para tanto, foi realizada uma transfecção em células HEK293T (aderidas) com o vetor pcDNA 3.4-TOPO. Foi obtida uma expressão de 21,26 ± 8,3 μg/mL de hPRL no meio condicionado sem soro. A hPRL foi purificada por cromatografia de afinidade a metais imobilizados (IMAC), eluindo 92% da hPRL em uma única fração que, analisada por HPSEC, apresentou pureza de 97%. O perfil de N-glicanos da amostra apresentou seis espécies, todas com terminação em ácido-siálico, do tipo complexo, sendo bi, tri e tetra-antenárias, com relativa predominância da espécie N2G2S1 (29,4%). A bioatividade in vitro da G-hPRL HEK293 demonstrou ser ≅ 16 vezes menor que a G-hPRL produzida em células CHO. / Human prolactin (hPRL) is a hormone synthesized by the pituitary gland with innumerable functions such as lactation, reproduction and osmotic regulation. This hormone is often determined in cases of lactation problems, infertility, and studies that elucidate its connection in some types of cancer (breast, prostate and uterus). The hPRL is found in the non-glycosylated (NG-hPRL) (23 kDa) and glycosylated (G-hPRL) (25 kDa) form, being the glycosylated isoform an ideal model for N-glycan profile analysis, since it has a single glycosylation site located in Asparagine 31. Glycosylation is directly related to solubility, stability, folding, half-life and biological activity in vivo. Chinese hamster ovary (CHO) cells and human embryonic kidney (HEK293) cells are the most widely used hosts for expression of recombinant proteins, since they can be grown at high densities and have similarity in post-translational modifications. The objective of this work was to express, purify and perform a physicochemical and biological characterization of the glycosylated hPRL from HEK293 cells, including analysis of the carbohydrate structure. For this purpose, a transfection was performed on HEK293T (adhered) cells with the 3.4-TOPO pcDNA vector. Expression of 21.26 ± 8.3 μg/mL hPRL in the serum free conditioned medium was obtained. The hPRL was purified by immobilized metal affinity chromatography (IMAC), eluting 92% of the hPRL in a single fraction which analyzed by HPSEC, showed 97% purity. The N-glycans profile of the sample showed six species, all with sialic acid termination, complex type, being bi, tri and tetra antennary, with a relative predominance of N2G2S1 (29.4%). In vitro bioactivity of G-hPRL HEK293 demonstrated to be ≅ 16-fold lower than G-hPRL produced in CHO cells.

O transplante de ilhotas pancreáticas é uma terapia promissora para o tratamento da diabetes mellitus tipo 1 (DM1). No entanto, ilhotas transplantadas estão sujeitas à rejeição pelo sistema imune dos pacientes receptores, portanto faz-se necessário o desenvolvimento de mecanismos moleculares que protejam essas células. Estudos mostraram que o hormônio prolactina (PRL) é capaz de inibir a apoptose desencadeada por citocinas pró-inflamatórias sobre células beta pancreáticas e que este processo citoprotetor depende da presença da chaperona HSPB1. Foi observado que durante o desenvolvimento do DM1, as células beta pancreáticas sofrem estresse de retículo endoplasmático e que isso contribui para desencadear apoptose. O estresse de retículo endoplasmático é caracterizado pelo acúmulo de proteínas mal dobradas nessa organela resultando na ativação da resposta a proteínas mal dobradas (UPR) que tem como finalidade recuperar a homeostase celular. No presente estudo mostramos, pela primeira vez, que PRL foi capaz de proteger células beta pancreáticas contra estresse de retículo endoplasmático promovido tanto por citocinas pró-inflamatórias (TNFα, IFNγ e IL1β) quanto pelos estressores de retículo endoplasmático: tunicamicina e tapsigargina; e que HSPB1 é essencial nesse mecanismo de citoproteção. No contexto do DM1, esse hormônio parece ter um efeito modulador da UPR aumentando os níveis de BiP, antecipando a ativação de ATF6 e PERK, mantendo a via de PERK ativa por mais tempo, inibindo a via de IRE1α, e diminuindo os níveis de CHOP em tempos maiores. Coletivamente, os resultados aqui apresentados aprofundam os conhecimentos sobre a função de HSPB1, conduzindo para o desenvolvimento de estratégias que visam à atenuação da morte de células beta por meio da modulação de uma via de proteção endógena, a qual é independente da modulação do sistema imunológico. / The islet transplantation is a promising therapy for the treatment of type 1 diabetes mellitus (T1DM). However, transplanted islets are subject to rejection by the immune system of the recipient patients, therefore the development of molecular mechanisms that protect these cells is necessary. Studies have shown that the hormone prolactin (PRL) is capable of inhibiting apoptosis triggered by pro-inflammatory cytokines on pancreatic beta cells and that this cytoprotective process depends on the presence of the chaperone HSPB1. It was observed that during the development of type 1 diabetes, pancreatic beta cells undergo endoplasmic reticulum stress and that this contributes to trigger apoptosis. The endoplasmic reticulum stress is characterized by accumulation of misfolded proteins in this organelle resulting in the activation of unfolded protein response (UPR) that aims to restore cellular homeostasis. In the present study, we show for the first time that PRL was able to protect pancreatic beta cells against endoplasmic reticulum stress promoted by both pro-inflammatory cytokines (TNFα, IFNγ and IL1β) as the endoplasmic reticulum stressors: tunicamycin and thapsigargin; and HSPB1 is essential that cytoprotective mechanism. In the context of T1DM, PRL appears to have a modulating effect of the UPR by increasing the levels of BiP, anticipating the activation of ATF6 and PERK, keeping the PERK pathway active for longer, inhibiting the pathway IRE1α, and decreasing the levels of CHOP for longer times. Collectively, the results presented here deepen the knowledge of the HSPB1 function, leading to the development of strategies inducing attenuation of beta cells death through modulation of endogenous protection means, which are independent of the modulation of the immune system.