Thyroid hormone is a master regulator of growth and development in all vertebrates. Thus, disruption of its synthesis and activity can lead to profound consequences. Past decade studies on thyroid function tests have established an efficient guideline for monitoring thyroid diseases, yet a significant proportion of healthcare providers do not defer to it in their practice. The aim of this study is to assess provider practices in the diagnosis and treatment of primary hypothyroidism due to autoimmunity at Boston Children's Hospital (CHB) for a primarily pediatric patient population. Commonly known as Hashimoto's thyroiditis (HT), this is the most common thyroid disease in the world as well as the most common manifestation of human autoimmune endocrine disease. Through CHB's bioinformatics institute, a rich data set was collected to assess the manner in which healthcare providers utilized relevant thyroid function tests (TFTs). This work assessed and confirmed the superior sensitivity of thyroid peroxidase autoantibodies (TPO) relative to thyroglobulin antibodies (TgAb) for diagnosing HT in children. We also verified proper utilization of thyroid stimulating hormone tests to monitor HT but concluded that there is a low utilization efficiency with regards to measurements of thyroid hormones (thyroxine and triiodothyronine). Based upon the observation of unnecessary monetary loss caused by improper TFTs utilization, it can be concluded that reflex testing at CHB may improve provider practices' efficiency for HT monitoring.

<p>Polybrominated diphenyl ethers (PBDEs) are a class of brominated flame retardant chemicals that are added to plastics, electronic components, furniture foam, and textiles to reduce their combustibility. Of the three commercial mixtures historically marketed, only DecaBDE, which is constituted almost entirely (~97%) of the fully brominated congener decabromodiphenyl ether (BDE-209), continues to be used in the U.S. today. While decaBDE is scheduled for phase-out in the U.S. at the end of 2013, exposures to BDE-209 and other PBDEs will continue into the foreseeable future as products that contain them continue to be used, recycled, and discarded. In addition, decaBDE use continues to be largely unrestricted across Asia, although restricted from use in electronic equipment in Europe. </p><p>Despite limits placed on PBDE uses, they are ubiquitous contaminants detected worldwide in humans and wildlife. Major health effect concerns for PBDEs come largely from evidence in laboratory rodents demonstrating neurotoxicity, reproductive and developmental impairments, and thyroid disruption. The potential for PBDEs, particularly BDE-209, to disrupt thyroid regulation and elicit other toxic outcomes in fish is less clear. Thus, the overall objective of this thesis research was to answer questions concerning how fish, as important indicators of overall environmental health, are metabolizing PBDEs and whether and how PBDEs are disrupting thyroid hormone regulation. The central hypothesis was that PBDE metabolism in fish is mediated by iodothyronine deiodinase (dio) enzymes, which are responsible for activating and inactivating thyroid hormones, and that PBDE exposures are causing thyroid system dysfunction across fish life stages. </p><p>Under the first research aim, in vitro experiments conducted in liver tissues isolated from common carp (Cyprinus carpio) suggested a role for dio enzymes in catalyzing the reductive debromination of PBDEs. Carp liver microsomes efficiently debrominated BDE-99 to BDE-47, and enzymes catalyzing this reaction were associated predominantly with the endoplasmic reticulum (i.e., microsomal fraction) where dio enzymes are located. Competitive substrate experiments in carp liver microsomes also demonstrated that rates of BDE-99 debromination to BDE-47 were significantly inhibited upon challenges with 3,3',5'-triiodothyronine (rT3) and thyroxine (T4). This finding supported the hypothesis that enzymes involved in the metabolism of PBDEs may have high affinities for thyroid hormones. Indeed, experiments to determine apparent enzymatic kinetics (apparent Vmax and Km values) of BDE-99 hepatic metabolism suggested that enzymes responsible for the catalytic activity appeared to have a higher affinity for native thyroid hormone than BDE-99. </p><p>The second and third research aims were focused on evaluating BDE-209 accumulation, metabolism, and thyroid toxicity in juvenile and adult life stages of fish using the fathead minnow (Pimephales promelas) as a model. BDE-209 bioaccumulated and was debrominated to several reductive metabolites ranging from penta- to octaBDEs in both juvenile and adult fish exposed to BDE-209. In addition, thyroid hormone regulation in juvenile and adult male fathead minnows was severely disrupted by BDE-209 at low, environmentally relevant exposures. In juvenile minnows, the activity of dio enzymes (T4-outer ring deiodination; T4-ORD and T4-inner ring deiodination; T4-IRD) declined by ~74% upon oral doses of 9.8 ± 0.2 µg/g wet weight (ww) food at 3% body weight (bw)/day for 28 days, compared to controls. Declines in dio activity were accompanied by thyroid follicle hypertrophy indicative of over-stimulation and injury. In addition to thyroid disruption, a distinctive liver phenotype characterized by vacuolated hepatocyte nuclei was measured in ~48% of hepatocytes from treated fish that was not observed in controls. </p><p>Under the third research aim, adult male fathead minnows received dietary treatments of BDE-209 at a low dose (95.3 ± 0.41 ng/g-food at 3% bw/day) and a high dose (10.1 ± 0.10 µg/g-food at 3% bw/day) for 28 days followed by a 14-day depuration period to evaluate recovery. Compared to negative controls, adult male fish exposed orally to BDE-209 at the low dose tested for 28 days experienced a 53% and 46% decline in circulating total T4 and T3, respectively, while fish at the high BDE-209 dose tested had total T4 and T3 deficits of 59% and 62%, respectively. Depressed levels of plasma thyroid hormones were accompanied by a 45-50% decline in the rate of T4-ORD in brains of all treatments by day 14 of the exposure. The decreased T4-ORD continued in the brain at day 28 with a ~65% decline measured at both BDE-209 doses. BDE-209 exposures also caused transient, tissue-specific upregulations of relative mRNA transcripts encoding dio enzymes (dio1, dio2), thyroid hormone receptors (TR&alpha, TR&beta), and thyroid hormone transporters (MCT8, OATP1c1) in the brain and liver in patterns that varied with time and dose, possibly as a compensatory response to hypothyroidism. In addition, thyroid perturbations at the low dose tested generally were equal to those measured at the high dose tested, suggesting non-linear relationships between PBDE exposures and thyroid dysfunction in adult fish. Thus, mechanisms for BDE-209 induced disruption of thyroid regulation can be proposed in adult male minnows that involve altered patterns of thyroid hormone signaling at several important steps in their transport and activation. </p><p>A growing body of evidence describing PBDE toxicity in biota, including data generated here, along with studies showing continued and rising PBDE body burdens, raises concern for human and wildlife health. Long delays in removing PBDEs from the market, their ongoing presence in many products still in use, and their active use outside the U.S. and European Union will leave a lasting legacy of rising contamination unless more concerted regulatory and policy actions are taken to reduce future exposures and harm.</p> / Dissertation

Maturity-onset diabetes of the young (MODY), due to hepatocyte nuclear factor 1 alpha mutations (HNF1A-MODY), is the most common form of monogenic diabetes presenting in young adults. An accurate genetic diagnosis of HNF1A-MODY has therapeutic implications for the patients and their family members. However, the majority of people with HNF1A-MODY are not referred for genetic testing and remain misdiagnosed as type 1 or type 2 diabetes. As part of measures to address this misdiagnosis, over the last few years there have been efforts to define clinical features and biomarkers that can be used to identify those at high risk of HNF1A-MODY. Secreted hepatic proteins regulated by HNF1A are attractive candidates for diagnostic biomarkers that would be specific for this form of diabetes. Apolipoprotein M (apoM), C-reactive protein (CRP) and plasma glycan profile have all been investigated as biomarkers to improve selection of suspected MODY cases for genetic testing. In my thesis, I have addressed questions about the variation in apoM between different forms of diabetes and assessed the performance of hsCRP and plasma glycan profile to identify HNF1A-MODY in previously uninvestigated individuals with young-onset diabetes and in a non-European population. Additionally because CRP and plasma glycans are both important components of an acute inflammatory response, I examined the effect of haploinsufficiency of HNF1A in a standardised model of inflammation. When investigating apoM, I showed that serum apoM levels are lower in HNF1A-MODY than controls, and have demonstrated for the first time that serum apoM provides good discrimination between HNF1A-MODY and type 1 diabetes. CRP and plasma glycan profile both performed well in identifying HNF1A-MODY cases in unselected young adults with diabetes. The results also suggested that both biomarkers have value for assessing the functional impact of novel HNF1A variants. I went on to examine the use of a low CRP for selecting those at risk of HNF1A-MODY in South Asian subjects with young-onset diabetes. This study suggests that the overall population prevalence of HNF1A-MODY is similar in South Asians to Europeans, but that MODY represents a lower proportion of those with diabetes (due to the higher prevalence of type 2 diabetes in South Asians). The specific selection strategy employed in this study was not successful in identifying subjects at high risk of HNF1A-MODY (only 3% of those sequenced had mutations), suggesting that additional clinical and biochemical features will be required in addition to CRP to distinguish South Asians at high risk of HNF1A-MODY. Lastly, using endotoxaemia as a standardised model of acute inflammation for the first time in HNF1A-MODY, I have shown that despite low baseline levels, subjects with HNF1A-MODY had peak stimulated CRP levels comparable to non-diabetic controls. An attenuated cytokine response was observed in HNF1A-MODY, which requires further investigation. This is also the first report of inflammation-associated changes in plasma and white cell membrane glycan profile in diabetes. This research work adds substantially to current understanding of performance of HNF1A-MODY biomarkers, a critical step before their clinical translation. The work presented also provides novel insights into the regulation of the acute inflammatory response in HNF1A-MODY.

Posttraumatic stress disorder (PTSD) is a psychiatric condition affecting approximately 8% of the adult U.S. population with rates twice as high in women than men. Increasingly, evidence has suggested a close relationship between PTSD and increased risk of metabolic diseases. However, the literature on PTSD and metabolic disease risk factors has been limited by the lack of investigation of the potential influence of race on this relation. The current study examined the possible effect of race on the relation between PTSD and metabolic risk. Data for this study were provided from sample of that included 50 African American women and 39 Caucasian women, 56.2% and 43.8% respectively. Results support the importance of race in the relationship between PTSD and metabolic disease risk factors. Future research would benefit from analysis of cultural factors to explain how race might influence the course of metabolic disease risk and development in women with PTSD.

The discovery that bile acids act as endogenous ligands of the membrane receptor TGR5 and the nuclear receptor FXR increased their significance as regulators of cholesterol, glucose and energy metabolism. Activation of TGR5, expressed on enteroendocrine L cells, by bile acids caused secretion of GLP-1, which stimulates insulin secretion from pancreatic β cells. Expression of TGR5 on pancreatic islet cells and the direct effect of bile acids on the endocrine functions of pancreas, however, are not fully understood. The aim of this study was to identify expression of TGR5 in pancreatic islet cells and determine the effect of bile acids on insulin secretion. Expression of TGR5 was identified by quantitative PCR and western blot in islets from human and mouse, and in α (αTC1-6) and β (MIN6) cells. Release of insulin, glucagon and GLP-1 were measured by ELISA. The signaling pathways coupled to TGR5 activation were identified by direct measurements such as stimulation of G proteins, adenylyl cyclase activity, PI hydrolysis and intracellular Ca2+ in response to bile acids; and confirmed by the use of selective inhibitors that block specific steps in the signaling pathway. Our studies identified expression of TGR5 receptors in β cells and demonstrated that activation of these receptors by both pharmacological ligands (oleanolic acid (OA) and INT-777) and physiological ligand (lithocholic acid, LCA) induced insulin secretion. TGR5 receptors are also expressed in α cells and, activation of TGR5 by OA, INT-777 and LCA at 5 mM glucose induced release of glucagon, which is processed from proglucagon by the selective expression of prohormone convertase 2 (PC2). However, under hyperglycemia, activation of TGR5 in α cells augmented the glucose-induced increase in GLP-1 secretion, which in turn, stimulated insulin secretion. Secretion of GLP-1 from α cells reflected TGR5-mediated increase in PC1 promoter activity and PC1 expression, which selectively converts proglucagon to GLP-1. The signaling pathway activated by TGR5 to mediate insulin and GLP-1 secretion involved Gs/cAMP/Epac/PLC-ε/Ca2+. These results provide insights into the mechanisms involved in the regulation of pancreatic α and β cell function by bile acids and may lead to new therapeutic avenues for the treatment of diabetes.

Dans un contexte mondial d’émergence de bactéries résistantes aux antibiotiques, il est nécessaire d’explorer de nouvelles voies de recherche pour trouver de nouveaux traitements. Cet état de fait est particulièrement marqué dans le cadre d’infections chroniques associées à une colonisation des tissus par des biofilms bactériens. L’endocrinologie microbienne est un champ de recherche axé sur l’étude des mécanismes de communication inter-règnes qui peut s’établir entre des bactéries et leurs hôtes. Les molécules humaines qui permettent ce dialogue constituent de potentiels outils capables de moduler la physiologie des bactéries pour empêcher leur développement. Dans cette optique, l’objectif de ma thèse était d’approfondir nosconnaissances sur l’effet des peptides natriurétiques, une famille d’hormones humaines, sur la physiologie du pathogène opportuniste P. aeruginosa. Les travaux que j’ai menés ont permis de préciser les mécanismes de l’action anti-biofilm du peptide natriurétique de type C ou CNP. J’ai également montré qu’un autre peptide, le peptide natriurétique atrial (ANP), est capable de disperser un biofilm établis de P. aeruginosa. Dans un second temps, j’ai pu identifier que l’opéron ami, dans son intégralité, est indispensable aux effets des peptides natriurétiques et qu’en plus les protéines codées par les gènes de l’opéron ami ont un rôle important dans la régulation de la virulence bactérienne et dans la formation des biofilms. Ainsi, j’ai pu mettre en évidence que les protéines AmiE et AmiR, en plus de leur rôle dans le métabolisme secondaire, sont impliquées dans la régulation de la virulence et de la formation de biofilm de P. aeruginosa. / In a global context of emergence of antibiotic-resistant bacteria, it is necessary to explore new paths of research to find new treatments. This state of affairs is particularly marked in the context of chronic infections associated with tissue colonization by bacterial biofilms. Microbial endocrinology is a field of research focused on the study of inter-kingdom communication that can be established between bacteria and their hosts. The human molecules that allow this dialogue are potential tools capable of modulating bacterial physiology to prevent their development. In this perspective, the aim of my thesis was to deepen our knowledge about the effect of natriuretic peptides, a family of human hormones, on the physiology of the opportunistic pathogen P. aeruginosa. The work that I carried out allowed us to characterize the mechanisms of the anti-biofilm action of the natriuretic peptide type C or CNP. I have also shown that another peptide, the atrial natriuretic peptide (ANP), is able to disperse an established biofilm of P. aeruginosa. In a second step, I was able to identify that the entire ami operon is essential for the effects of the natriuretic peptides and that the proteins encoded by the genes of the ami operon have an important role in bacterial virulence regulation and in the formation of biofilms. Thus, I was able to demonstrate that the AmiE and AmiR proteins, in addition to their role in secondary metabolism, are involved in the regulation of virulence and biofilm formation of P. aeruginosa.

Introdução: O sequenciamento completo do genoma humano foi finalizado em 2003, tornando-se responsável por uma revolução na prática médica, geralmente referenciada como a era da genômica e da medicina personalizada. O aumento na demanda por testes genéticos e a introdução de novos métodos de sequenciamento em larga escala tem gerado um inevitável crescimento no número de variantes raras mapeadas. Tal fato levou à expansão de áreas do conhecimento necessárias, tanto para uma adequada avaliação do real significado clínico desses achados, como para uma indicação mais criteriosa da investigação genética. Devido a isso, torna-se necessário otimizar os processos, desde a seleção dos casos sob suspeita até a coleta, armazenamento e análise dos dados clínico-laboratoriais e moleculares obtidos, visando a uma melhor acurácia no diagnóstico, tratamento e aconselhamento genético. Uma obtenção e interpretação adequada dos achados genético-moleculares, aliados a critérios clínicos de seleção adequados, resultaria no que hoje constitui a análise genética multifatorial ou integrada. Objetivo: O presente trabalho teve como objetivo implementar a análise genético-molecular integrada ao processo de investigação de pacientes com diagnóstico clínico de MODY (Maturity-Onset Diabetes of the Young). Materiais e métodos: A prevalência dos dois subtipos mais frequentes de MODY (MODY-GCK e MODY-HNF1A) foi investigada em uma grande coorte de famílias brasileiras. Todas as variantes identificadas como relacionadas ao fenótipo foram criteriosamente classificadas quanto a suas reais evidências de patogenicidade por meio da implementação das mais recentes diretrizes de obtenção e interpretação de achados genéticos. Resultados: Cento e nove probandos foram investigados, 45% (49/109) com suspeita clínica de MODYGCK e 55% (60/109) de MODY-HNF1A, além de 94 familiares, o que resultou na identificação de 25 variantes únicas candidatas no gene GCK em 30 probandos (61% - 30/49), além de 7 em 10 indivíduos com suspeita de MODYHNF1A (17% - 10/60). Dos 87 familiares sob risco rastreados, 43 eram portadores da mesma variante da família (37 - GCK e 6 - HNF1A). Um provável efeito fundador foi identificado com relação a uma deleção/inserção do tipo frameshift, presente em três probandos com MODY-GCK oriundos da mesma região do país. A implementação dos critérios da ACMG (The American College of Medical Genetics and Genomics) de avaliação de evidências de patogenicidade resultou na classificação de uma grande parcela das variantes como patogênica (36% - GCK / 86% - HNF1A) e provavelmente patogênica (44% - GCK / 14% - HNF1A), restando 16% com uma associação ainda incerta com o fenótipo investigado. Quatorze novas variantes foram identificadas (12 - GCK / 2 - HNF1A), ampliando, assim, o espectro de alterações associadas a MODY. Conclusões: Esta abordagem investigativa nos permitiu não apenas esclarecer a etiologia genética de inúmeros casos com diagnóstico clínico de MODY, como também determinar a classificação de patogenicidade das variantes de uma maneira mais detalhada, reforçando, principalmente, a provável relação com o fenótipo dentre aquelas ainda não descritas / Introduction: The full sequencing of the human genome was completed in 2003, becoming responsible for a revolution in medical practice, generally referred to as the era of genomics and personalized medicine. The increase in demand for genetic testing and the introduction of new methods of large-scale sequencing has raised an inevitable growth in the number of mapped rare variants. This fact led to the expansion of areas of knowledge required for a proper evaluation of the real clinical significance of these findings, as to a more solid indication of genetic research. Because of this, it is necessary to optimize the processes, from the selection of cases under suspicion until the collection, storage and analysis of the clinical laboratory and molecular data obtained, aiming a better accuracy in diagnosis, treatment and genetic counseling. A proper collection and interpretation of the molecular genetic findings, coupled with clinical criteria for appropriate selection would result in what today is the multifactorial or integrated genetic analysis. Objective: This current study aimed to implement the integrated molecular genetic analysis to the investigation process of patients with clinical diagnosis of MODY (Maturity Onset Diabetes of the Young). Materials and methods: The prevalence of the two most common MODY subtypes (MODYGCK and MODY-HNF1A) was investigated in a large cohort of Brazilian families. All variants identified as related to the phenotype were carefully classified to their actual evidence of pathogenicity by implementing the latest guidelines for obtainment and interpretation of genetic findings. Results: 109 probands were investigated, 45% (49/109) with clinical suspicion of MODY-GCK and 55% (60/109) of MODY-HNF1A, plus 94 family members under risk, which resulted in the identification of 25 unique variants candidates in the gene GCK in 30 probands (61% - 30/49), and also 7 in 10 individuals with suspected MODYxvi HNF1A (17% - 10/60). Of the 87 family members under risk, 43 were carriers of the same variant of the family (37 - GCK and 6 - HNF1A). A probable founding effect was identified, related to a frameshift deletion/insertion, present in three probands with MODY-GCK from the same region of the country. The implementation of the ACMG guidelines (The American College of Medical Genetics and Genomics) of evidence of pathogenicity assessment resulted in the classification of a large portion of the variants as pathogenic (36% - GCK / 86% - HNF1A) and likely pathogenic (44% - GCK / 14% - HNF1A), leaving 16% with a still uncertain association with the investigated phenotype. Fourteen new variants were identified (12 - GCK / 2 - HNF1A), broadening the spectrum of modifications associated to MODY. Conclusions: This investigative approach allowed us to not only clarify the genetic etiology of numerous cases with clinical diagnosis of MODY, as well as determine the pathogenicity classification of variants in a more detailed way, reinforcing the likely relationship with the phenotype among those not yet described

Devido as atuais mudanças globais é esperado que um grande número de espécies necessitem de uma integração de ações dentro e fora do seu ambiente natural para a conservação. O bioma cerrado é um dos ecossistemas mais ricos em biodiversidade, mas devido a ação antrópica, resta menos de 20% de sua cobertura vegetal original. A Raposinha do Campo (Lycalopex vetulus) é um canídeo de pequeno porte (2,5-4kg) endêmico do cerrado do Brasil central e está presente na lista dos animais ameaçados de extinção do Estado de São Paulo. Não existe em literatura uma descrição detalhada do ciclo estral desta espécie. O presente estudo teve como objetivo caracterizar a ciclicidade ovariana de Lycalopex vetulus com o uso de método não-invasivo: Extração e mensuração de metabólitos fecais de progesterona e estradiol, com o uso da técnica de enzimaimunoensaio, verificando também a possível existência de diferenças mensais entre as médias das concentrações dos referidos metabólitos em um período de 12 meses. Foram utilizadas 8 fêmeas, adultas, presentes em cinco instituições no estado de São Paulo, onde foram coletadas fezes 3 vezes por semana de cada indivíduo, durante 12 meses, para a extração e mensuração dos metabólitos de progesterona, estradiol e corticosterona. Os perfis das excreções dos metabólitos fecais dos hormônios sexuais de 6 das 8 fêmeas, foram muito semelhantes, sendo período de maior atividade reprodutiva entre os messes de julho, agosto e setembro, ocorrendo mais de um ciclo ovulatório dentro desse período. Nos outros meses do ano, apesar de algumas variações acima da linha basal, não foram encontrados indícios de atividade reprodutiva. De acordo com essas características, semelhante entre a maioria das fêmeas do estudo, podemos sugerir que a raposa-do-campo seja poliestrica sazonal, com atividade reprodutiva ocorrendo nos meses de julho, agosto e setembro. Esses achados são muito próximos do visualizado em populações de vida livre, onde acasalamentos são observados entre junho e julho. O perfil de excreção dos metabólitos fecais de glicocorticóides segue o mesmo padrão observado para os metabólitos de progesterona e estradiol, com um aumento mais significativo da excreção nos meses de julho, agosto e setembro. Esses resultados indicam que além da grande quantidade de estressores que podem alterar a excreção de glicocorticóides, também deve-se considerar a flutuação sazonal e o status reprodutivo do indivíduo ao avaliar as concentrações de metabólitos de glicocorticóides. Duas fêmeas, que dividem o mesmo recinto, não apresentaram um padrão de ciclicidade reprodutiva. Os resultados obtidos nesse estudo indicam que a dosagem de metabólitos fecais de progesterona e estradiol podem ser usadas para diferenciar o período reprodutivo do período não reprodutivo em fêmeas de Lycalopex vetulus, fornecendo informações importantes sobre a biologia reprodutiva da espécie, o que pode contribuir no desenvolvimento de estratégias para a conservação desta espécie, como por exemplo aumentar o sucesso reprodutivo ex situ. / Due to the current global changes is expected that a large number of species require an integration of actions inside and outside their natural environment for them conservation. The cerrado is one of the richest ecosystems in biodiversity, but due to human action, there remains less than 20% of its original vegetation cover. The Hoary fox (Lycalopex vetulus) is a small canid (2,5-4kg) endemic of the cerrado from the central Brazil and is present in the list of endangered species in the state of São Paulo. It was not found in literature any detailed description of the estrous cycle of this species. The present study aimed to characterize ovarian cyclicity in Lycalopex vetulus using a noninvasive method: Extraction and measurement of fecal metabolites of estradiol and progesterone, using the technique of enzymeimmunoassay, It was also verified the possible differences between the monthly mean concentrations of these metabolites in a period of 12 months. 8 captive adult females, were studied in five different institutions in the state of São Paulo, where feces were collected three times per week for each individual for 12 months for extraction and measurement of metabolites of progesterone, estradiol and glucocorticoids. The profile of the concentration of fecal metabolites of sexual hormones from 6 of 8 females was very similar, being the period of major reproductive activity between the months of July, August and September, with the occurrence of more than one ovulatory cycle within that period. In the others months of the year, despite some variations above the baseline, no evidence of reproductive activity were found. According to these characteristics, similar in most females of the study, we can suggest that the hoary fox is seasonal polyestrous with reproductive activity occurring in the months of July, August and September. These findings are very close to the observed in wild populations, where mating is observed between June and July. The profile of excretion of fecal glucocorticoid metabolites follows the same pattern observed for the metabolites of progesterone and estradiol, with a more significant increase in excretion in the months of July, August and September. These results indicate that besides the large amount of stressors that can alter the excretion of glucocorticoids also it must be considered the seasonal fluctuation and the reproductive status of the individual to evaluate the levels of glucocorticoids. Two females, who share the same captivity, did not show a pattern of reproductive cyclicity. The results of this study indicate that the dosage of fecal metabolites of estradiol and progesterone can be used to differentiate the reproductive period of non-reproductive period in females of Lycalopex vetulus, providing important information about the reproductive biology, which may contribute to the development of the species conservation strategies, such as increasing the reproductive ex situ success.

Devido as atuais mudanças globais é esperado que um grande número de espécies necessitem de uma integração de ações dentro e fora do seu ambiente natural para a conservação. O bioma cerrado é um dos ecossistemas mais ricos em biodiversidade, mas devido a ação antrópica, resta menos de 20% de sua cobertura vegetal original. A Raposinha do Campo (Lycalopex vetulus) é um canídeo de pequeno porte (2,5-4kg) endêmico do cerrado do Brasil central e está presente na lista dos animais ameaçados de extinção do Estado de São Paulo. Não existe em literatura uma descrição detalhada do ciclo estral desta espécie. O presente estudo teve como objetivo caracterizar a ciclicidade ovariana de Lycalopex vetulus com o uso de método não-invasivo: Extração e mensuração de metabólitos fecais de progesterona e estradiol, com o uso da técnica de enzimaimunoensaio, verificando também a possível existência de diferenças mensais entre as médias das concentrações dos referidos metabólitos em um período de 12 meses. Foram utilizadas 8 fêmeas, adultas, presentes em cinco instituições no estado de São Paulo, onde foram coletadas fezes 3 vezes por semana de cada indivíduo, durante 12 meses, para a extração e mensuração dos metabólitos de progesterona, estradiol e corticosterona. Os perfis das excreções dos metabólitos fecais dos hormônios sexuais de 6 das 8 fêmeas, foram muito semelhantes, sendo período de maior atividade reprodutiva entre os messes de julho, agosto e setembro, ocorrendo mais de um ciclo ovulatório dentro desse período. Nos outros meses do ano, apesar de algumas variações acima da linha basal, não foram encontrados indícios de atividade reprodutiva. De acordo com essas características, semelhante entre a maioria das fêmeas do estudo, podemos sugerir que a raposa-do-campo seja poliestrica sazonal, com atividade reprodutiva ocorrendo nos meses de julho, agosto e setembro. Esses achados são muito próximos do visualizado em populações de vida livre, onde acasalamentos são observados entre junho e julho. O perfil de excreção dos metabólitos fecais de glicocorticóides segue o mesmo padrão observado para os metabólitos de progesterona e estradiol, com um aumento mais significativo da excreção nos meses de julho, agosto e setembro. Esses resultados indicam que além da grande quantidade de estressores que podem alterar a excreção de glicocorticóides, também deve-se considerar a flutuação sazonal e o status reprodutivo do indivíduo ao avaliar as concentrações de metabólitos de glicocorticóides. Duas fêmeas, que dividem o mesmo recinto, não apresentaram um padrão de ciclicidade reprodutiva. Os resultados obtidos nesse estudo indicam que a dosagem de metabólitos fecais de progesterona e estradiol podem ser usadas para diferenciar o período reprodutivo do período não reprodutivo em fêmeas de Lycalopex vetulus, fornecendo informações importantes sobre a biologia reprodutiva da espécie, o que pode contribuir no desenvolvimento de estratégias para a conservação desta espécie, como por exemplo aumentar o sucesso reprodutivo ex situ. / Due to the current global changes is expected that a large number of species require an integration of actions inside and outside their natural environment for them conservation. The cerrado is one of the richest ecosystems in biodiversity, but due to human action, there remains less than 20% of its original vegetation cover. The Hoary fox (Lycalopex vetulus) is a small canid (2,5-4kg) endemic of the cerrado from the central Brazil and is present in the list of endangered species in the state of São Paulo. It was not found in literature any detailed description of the estrous cycle of this species. The present study aimed to characterize ovarian cyclicity in Lycalopex vetulus using a noninvasive method: Extraction and measurement of fecal metabolites of estradiol and progesterone, using the technique of enzymeimmunoassay, It was also verified the possible differences between the monthly mean concentrations of these metabolites in a period of 12 months. 8 captive adult females, were studied in five different institutions in the state of São Paulo, where feces were collected three times per week for each individual for 12 months for extraction and measurement of metabolites of progesterone, estradiol and glucocorticoids. The profile of the concentration of fecal metabolites of sexual hormones from 6 of 8 females was very similar, being the period of major reproductive activity between the months of July, August and September, with the occurrence of more than one ovulatory cycle within that period. In the others months of the year, despite some variations above the baseline, no evidence of reproductive activity were found. According to these characteristics, similar in most females of the study, we can suggest that the hoary fox is seasonal polyestrous with reproductive activity occurring in the months of July, August and September. These findings are very close to the observed in wild populations, where mating is observed between June and July. The profile of excretion of fecal glucocorticoid metabolites follows the same pattern observed for the metabolites of progesterone and estradiol, with a more significant increase in excretion in the months of July, August and September. These results indicate that besides the large amount of stressors that can alter the excretion of glucocorticoids also it must be considered the seasonal fluctuation and the reproductive status of the individual to evaluate the levels of glucocorticoids. Two females, who share the same captivity, did not show a pattern of reproductive cyclicity. The results of this study indicate that the dosage of fecal metabolites of estradiol and progesterone can be used to differentiate the reproductive period of non-reproductive period in females of Lycalopex vetulus, providing important information about the reproductive biology, which may contribute to the development of the species conservation strategies, such as increasing the reproductive ex situ success.

Abstract available through The FASEB Journal.