The prepartum surge in fetal plasma cortisol, essential for the maturation of organs in mammals and the normal timing of parturition in some species, including sheep, may result from an increase in the molar ratio of adrenocorticotropin (ACTH) to pro-opiomelanocortin (POMC) in the fetal circulation. Related to this, the cleavage of POMC to ACTH by the enzyme, prohormone convertase 1 (PC1), may be influenced by corticotrophin releasing hormone (CRH) stimulation. Accumulating evidence suggests that the capacity of individual corticotrophs to process POMC to ACTH may vary and individual corticotrophs are differentially responsive to CRH. It is not known, however, if there are separate corticotroph subpopulations in the fetal sheep pituitary which can be identified by differential colocalisation of POMC, ACTH and the CRH receptor 1, CRHR₁, nor if changes in the relative proportions of such subpopulations play a role in the molecular mechanisms underlying the overall changes in pituitary function described previously during gestation and in response to suboptimal uterine environments. To investigate these hypotheses, it was first necessary to develop novel methods for the simultaneous immunohistochemical labelling of POMC, ACTH and CRHR₁ in individual cells on sections of fetal sheep pituitary. In addition, I developed and validated an automated method to categorise and count individual cells to increase the quantitative power of this study. Pituitary tissue was collected from control fetuses at 53-55 (n=6), 63-85 (n=6), 110 (n=4), 139-141 (n=4) and 144-145 (n=6) days gestation. Two animal models, known to alter pituitary function in the fetal sheep, were used to investigate corticotrophic adaptations to suboptimal uterine environments. For the maternal periconceptional undernutrition (PCUN) model, maternal feed was reduced to 70% of maintenance requirements from at least 45 days before to 7 days after mating and fetal tissues were collected at 53-55 days gestation (n=7). For the placental restriction (PR) model, the majority of the placental attachment sites were removed in five ewes before mating and fetal tissues were collected at 140 (n=4) and 144 (n=4) days gestation. Pituitary sections were simultaneously labelled with antisera raised against full length POMC, ACTH and CRHR₁ and the proportions of pituitary cells with combinations of antisera were quantified. Four subpopulations of corticotrophs were identified, which expressed either: POMC+ACTH+CRHR₁, ACTH+CRHR₁, POMC+ CRHR₁ or POMConly. There was a significant decrease in the proportion of pituitary cells expressing POMC+ACTH+CRHR₁ between 53-55 and 65-85 days gestation, before an increase at 110 days gestation and a further marked decrease between 139-141 and 144-145 days gestation. In fetuses from the PCUN group, the proportion of pituitary cells expressing POMC+ACTH+CRHR₁ in early gestation was reduced. PR resulted in a significantly higher proportion of corticotrophs expressing POMC+ACTH+CRHR₁ during the prepartum period. This work represents the discovery of the differential expression of POMC, ACTH and CRHR₁ in individual corticotrophs of the fetal sheep pituitary and the first insights into the pituitary adaptations to periconceptional nutrient restriction and placental restriction at the level of individual corticotrophs. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1337370 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008

Coronary heart disease is the single greatest contributor to mortality within developed countries in our time. Type 2 diabetes mellitus and obesity, both of which are potent risk factors for coronary heart disease, are common conditions with rapidly rising rates of prevalence both globally and in Australia. Within Australia, Indigenous Australians suffer from disproportionately high rates of diabetes, obesity and coronary heart disease, the reason for which lies, in part, with changes in dietary intake and decreased levels of physical activity over the past 200 years. These disease trends have led to a substantially reduced life expectancy from an increase in excess cardiovascular deaths in Indigenous Australians. Assessment of cardiovascular risk in Indigenous Australians is complicated by ethnic differences in cardiovascular risk factors and their prevalence. There is also evidence that Indigenous Australians are genetically predisposed to diabetes. Current cardiovascular risk stratification algorithms are suboptimal, significantly underestimating risk in Indigenous Australians. This thesis aimed to address these issues. Firstly, to evaluate the efficacy of a lifestyle intervention programme to sustainably improve markers of health outcome in a cohort of Indigenous Australians who were overweight or had type 2 diabetes mellitus by improving diet and physical activity levels. Secondly, to evaluate the utility of anthropometric markers of obesity, ultrasound markers of atherosclerotic burden and serum C-reactive protein as additional cardiovascular risk stratification tools. The Healthy Lifestyle Programme (HELP) was a two year prospective intervention study designed and implemented with substantial input from the Indigenous community. The primary intervention consisted of a series of educational workshops delivered predominantly by Indigenous health workers advocating improving diet and increasing physical activity. To complement these workshops, facilities were established within the community to support dietary improvement and increased physical activity. Sustainable changes to markers of cardiovascular outcome, including significant reductions in central obesity and blood pressure were demonstrated over time. A slowing in the deterioration of renal function as marked by albuminuria was also detected. These beneficial changes were accompanied by increased physical activity and improved dietary intake. In a substudy, individuals with the apolipoprotein E4 genotype, who were dyslipidaemic at baseline, benefited from lipid profiles that improved significantly during the course of the study. Changes in blood pressure and central obesity detected during the course of the project are likely to have significant clinical impact on health outcomes considering their sustained nature. Evaluating ultrasound imaging based markers of cardiovascular risk, carotid artery intimal medial thickness was a more reliable measure of risk based on its firm correlation with established cardiovascular risk factors compared to brachial artery flow mediated vasodilatation. Carotid artery intimal medial thickness may be the better surrogate marker to improve cardiovascular risk stratification in the Indigenous population. Examining anthropometric markers of obesity, body mass index and waist circumference but not waist hip ratio correlated well with traditional cardiovascular risk factors. Furthermore, both body mass index and waist circumference predicted insulin resistance fairly well using threshold values derived from receiver operating characteristics curve analyses. Both body mass index and waist circumference were simple to perform with a high degree of reproducibility between observers and will likely be valuable additional cardiovascular risk stratification tools. Raised inflammatory stress, as marked by elevated serum C-reactive protein levels, was found in individuals with diabetes or obesity. C-reactive protein levels did not correlate with carotid artery intimal medial thickness, suggesting that C-reactive protein raises cardiovascular risk by means other than increasing atherosclerotic burden. Genotyping a small random sample of our the C-reactive protein gene promoter region. This SNP was found to independently increase serum C-reactive protein in the Framingham cohort. A genetic basis for increased inflammatory stress may contribute to coronary heart disease in Indigenous Australians. In summary, a community centred lifestyle intervention programme was efficacious in producing sustainable improvement in markers of health outcome. Examination of additional cardiovascular risk markers in the Indigenous community have provided evidence for using carotid artery intimal medial thickness, waist circumference, body mass index and serum C-reactive protein levels to improve cardiovascular risk stratification. These additional tools would allow for more specific resource allocation to target individuals at highest cardiovascular risk for preventative management. C-reactive protein gene polymorphisms, their contribution to circulating C-reactive protein levels and cardiovascular outcomes warrants further investigation in the Indigenous population. A more proactive risk management plan, which includes a lifestyle intervention component, may yield the greatest improvement in long term health outcomes if targeted at younger members of the Indigenous community.

Over the past decade, there has been unprecedented increase in the number of genetic loci associating with type 2 diabetes (T2D) risk and related glycemic traits, thanks to advances in sequencing technologies and access to large sample sizes. Identification of associated genetic variants across the frequency spectrum can provide valuable insight into disease pathophysiology. However, the translation into biological insights has been slow often due to uncertainties over the underlying effector transcripts. G6PC2/ABCB11 is one locus characterised by common non-coding variants that are strongly associated with fasting plasma glucose (FG) levels in healthy adults. The work presented in this thesis aims to understand how protein-coding variants in glycemic trait loci such as G6PC2 contribute to the variability of glycemic traits and in addition gain further insight into the physiological role of G6PC2. To evaluate the role of coding variants in glycemic trait variation, an exome array genotyping study of non-diabetic European individuals (n=33,407) reported multiple coding variants in G6PC2 that were independently associated with FG. I designed and conducted in vitro assays to functionally assess these variants and showed that they result in loss of function (LOF) due to reduced protein stability. This established G6PC2 as the effector transcript influencing FG and highlighted a critical role for G6PC2 (encoding the islet-specific glucose-6-phosphatase catalytic subunit) in glucose homeostasis. To investigate the role of low frequency (MAF=1-5%) and rare (MAF<1%) coding variants in influencing glycemic traits, recent large-scale exome array meta-analyses and whole exome sequencing were carried out as part of MAGIC (n=144,060) and the T2D-GENES/GoT2D consortia (n=12,940) respectively. G6PC1, a gene homolog of G6PC2 that primarily acts through the liver, was uncovered as a novel glycemic locus. My functional follow-up studies demonstrated that rare coding variants in G6PC1 exhibited LOF to influence both FG and FI levels. As rare variation in G6PC2 not previously identified could also affect G6PC2 function and modulate glycemic traits, I also functionally characterised a suite of rare non-synonymous G6PC2 variants. Most of the variants tested exhibited markedly reduced protein levels and/or loss of glycosylation. Several variants were also found to impact on enzymatic activity through inactivating or activating mechanisms to influence FG levels. Finally, to gain better understanding of the function of G6PC2 I performed gene knockdown studies in the EndoC-βH1 human beta cell model followed by insulin secretion analyses. G6PC2 knockdown resulted in increased insulin secretion at sub-threshold glucose stimulation levels, consistent with studies in knockout mouse models. In addition, expression of LOF G6PC2 variants were found to upregulate ER stress responses. These results warrant further studies of the precise roles that G6PC2, an ER-resident protein, plays in regulating insulin secretory function and ER homeostasis in the beta cell. Overall, my work described multiple rare coding variants in both G6PC1 and G6PC2 that alter protein function to regulate glucose metabolism through diverse mechanisms in different tissues. Improved understanding of these effector transcripts will open up opportunities for the exploration of new therapeutic targets for glucose regulation and T2D.

Durante as últimas duas décadas, as técnicas de análise de esteroides fecais têm sido utilizadas para pesquisa em endocrinologia reprodutiva em diversas espécies animais silvestres de vida livre, ou cativeiro. O presente estudo propõe a validação da técnica do ensaio imunoenzimático antiprogestinas CL425 e antiandrógenos R156/7 fecais em papagaio-de-peito-roxo (Amazona vinacea). Objetiva-se descrever os perfis endócrinos anuais dos esteroides sexuais urofecais em Amazona vinacea com potencial aplicação para o aprimoramento do manejo reprodutivo da espécie. Foram utilizados 10 casais adultos da espécie Amazona vinacea mantidos em viveiros suspensos, em Lomba Grande, Rio Grande do Sul, Brasil. As excretas foram coletadas duas vezes por semana, entre maio de 2012 e maio de 2013. O monitoramento da atividade gonadal foi feito por mensuração de metabólitos de andrógenos nas excretas dos machos e progestágenos, nas de fêmeas. Foram coletadas amostras frescas de excretas, consistentemente, entre 12h00min e 16h00min e mantidas congeladas até o processamento. O perfil anual de andrógenos nas excretas de machos de A. vinacea apresentou médias mensais variáveis de 25,9 ng/g, em janeiro até 125,7 ng/g, em setembro. O perfil anual de progestinas nas excretas de fêmeas A. vinacea, as médias mensais variaram de 2,76 ng/g, em dezembro até 45,7 ng/g, em setembro. Os perfis reprodutivos apresentaram variação na atividade hormonal, durante a fase reprodutiva. Porém, comparando-se casais reprodutores com não-reprodutores, observa-se que os níveis de progestágenos não diferiram significativamente entre fêmeas que fizeram postura e aquelas que não fizeram. Entretanto, os níveis de andrógenos diferiram significativamente entre reprodutores e não-reprodutores. Diferenças hormonais foram observadas nas diferentes estações anuais, tanto nos machos, quanto nas fêmeas. Dados metereológicos de temperatura e insolação demostraram correlação negativa com os perfís anuais de progestágenos. A utilização da metodologia aqui mencionada demonstrou aplicabilidade, praticidade e segurança como ferramenta para o estudo da fisiologia e do manejo reprodutivos de Amazona vinacea. / During the last two decades, the techniques of analysis of fecal steroids have been applied for studying reproductive profiles in many species of captive wild and free living wild animals. The advantages of this methodology are widely understood. This study aimed to validate the anti- CL425 progestin and anti-R156/7 androgen enzyme immunoassay in fecal samples from parrot-breasted-purple (Amazon vinacea). This communication describes the annual endocrine profiles of urofecal sex steroids in Amazona vinacea with potential application for improving the species reproductive management. In total, 10 adult A. vinacea pairs kept in suspended aviaries in southern Brazil were studied. Excreta samples were collected twice weekly between May 2012 and May 2013. Gonadal activity was monitored by measurement of androgen and progestin metabolites in excreta of birds. Fresh excreta samples were collected always between 12:00 and 16:00 hs, and kept frozen until processing. The samples were dried at 57oC, hormones crushed and extracted using 80% methanol. The hormone dosage was performed at the Laboratory of Endocrinology, NUPECCE (Center for Research and Conservation of Deer, Jaboticabal, SP) using enzyme immunoassay with antibody to androgen and progestin. Feces from 5 A. vinacea pairs were used in two distinct periods for assay validation, within and outside the breeding season. Tests included Parallelism, Dose-Response, and Physiological Validation. It was concluded that the enzyme immunoassay for determining progestin and androgen profiles in urofecal samples from A. vinacea is accurate, precise and reliable. The annual profile of androgen in excreta of A. vinacea resulted the following monthly averages: January 25.9 ± 3.7 ng/g (n = 90); February 28.7 ± 3.2 ng/g (n = 60); March 31 7 ± 17.7 ng/g (n = 80); April 27.9 ± 5.5 ng/g (n = 90); May 43.3 ± 7.9 ng/g (n = 90); June 49.1 ± 15.9 ng/g (n = 80); July 36.5 ± 7.5 ng/g (n = 90); August 86.9 ± 46.2 ng/g (n = 90); September 42 125.7 ± 1 ng/g (n = 80); October 110.3 ± 63.2 ng/g (n = 90); November 74.2 ± 57.7 ng/g (n = 90) and December 43.3 ± 13 4 ng/g (n = 80). The monthly averages in the annual profile of progestin in excreta of A. vinacea were as follows: January 7.2 ± 0.9 ng/g (n = 90); February 9.26 ± 1.3 ng/g (n = 60); March 15 8 ± 2.9 ng/g (n = 80); April 14.15 ± 1.85 ng/g (n = 90); May 16.05 ± 3.16 ng/g (n = 90); June 11.85 ± 2.54 ng/g (n = 80); July 11.89 ± 2.17 ng/g (n = 90); August 13.52 ± 3.48 ng/g (n = 90); September 55 ± 45.7 ng/g (n = 80); October 15.44 ± 24.91 ng/g (n = 90); November 4.27 ± 6.42 ng/g (n = 90) and December 2.76 ± 0.42 ng/g (n = 80). Reproductive profiles showed variation in hormonal activity during the reproductive phase. When comparing breeding to non-breeding pairs, it was observed that the progestin levels did not differ significantly between laying and not-laying birds. However, the androgen levels differed significantly between breeding and non-breeding birds. In addition, hormonal differences were observed in both male and female birds in accordance with different annual seasons. Meteorological temperature and insolation data showed a negative correlation with the annual progestin profiles. Measurement of androgen and progestin metabolites in A. vinacea feces proved to be a practical and safe tool for studying reproductive physiology and management of the species.

abstract: Type 1 diabetes (T1D) is a chronic disease that affects 1.25 million people in the United States. There is no known cure and patients must self-manage the disease to avoid complications resulting from blood glucose (BG) excursions. Patients are more likely to adhere to treatments when they incorporate lifestyle preferences. Current technologies that assist patients fail to consider two factors that are known to affect BG: exercise and alcohol. The hypothesis is postprandial blood glucose levels of adult patients with T1D can be improved by providing insulin bolus or carbohydrate recommendations that account for meal and alcohol carbohydrates, glycemic excursion, and planned exercise. I propose an evidence-based decision support tool, iDECIDE, to make recommendations to improve glucose control by taking into account meal and alcohol carbohydrates, glycemic excursion and planned exercise. iDECIDE is deployed as a low-cost and easy to disseminate smartphone application. A literature review was conducted on T1D and the state-of-the-art in diabetes technology. To better understand self-management behaviors and guide the development of iDECIDE, several data sources were collected and analyzed: surveys, insulin pump paired with glucose monitoring, and self-tracking of exercise and alcohol. The analysis showed variability in compensation techniques for exercise and alcohol and that patients made unaided decisions, suggesting a need for better decision support. The iDECIDE algorithm can make insulin and carbohydrate recommendations. Since there were no existing in-silico methods for assessing bolus calculators, like iDECIDE, I proposed a novel methodology to retrospectively compare insulin pump bolus calculators. Application of the methodology shows that iDECIDE outperformed the Medtronic insulin pump bolus calculator and could have improved glucose control. This work makes contributions to diabetes technology researchers, clinicians and patients. The iDECIDE app provides patients easy access to a decision support tool that can improve glucose control. The study of behaviors from diabetes technology and self-report patient data can inform clinicians and the design of future technologies and bedside tools that integrate patient’s behaviors and perceptions. The comparison methodology provides a means for clinical informatics researchers to identify and retrospectively test promising insulin blousing algorithms using real-life data. / Dissertation/Thesis / Doctoral Dissertation Biomedical Informatics 2017

Durante as últimas duas décadas, as técnicas de análise de esteroides fecais têm sido utilizadas para pesquisa em endocrinologia reprodutiva em diversas espécies animais silvestres de vida livre, ou cativeiro. O presente estudo propõe a validação da técnica do ensaio imunoenzimático antiprogestinas CL425 e antiandrógenos R156/7 fecais em papagaio-de-peito-roxo (Amazona vinacea). Objetiva-se descrever os perfis endócrinos anuais dos esteroides sexuais urofecais em Amazona vinacea com potencial aplicação para o aprimoramento do manejo reprodutivo da espécie. Foram utilizados 10 casais adultos da espécie Amazona vinacea mantidos em viveiros suspensos, em Lomba Grande, Rio Grande do Sul, Brasil. As excretas foram coletadas duas vezes por semana, entre maio de 2012 e maio de 2013. O monitoramento da atividade gonadal foi feito por mensuração de metabólitos de andrógenos nas excretas dos machos e progestágenos, nas de fêmeas. Foram coletadas amostras frescas de excretas, consistentemente, entre 12h00min e 16h00min e mantidas congeladas até o processamento. O perfil anual de andrógenos nas excretas de machos de A. vinacea apresentou médias mensais variáveis de 25,9 ng/g, em janeiro até 125,7 ng/g, em setembro. O perfil anual de progestinas nas excretas de fêmeas A. vinacea, as médias mensais variaram de 2,76 ng/g, em dezembro até 45,7 ng/g, em setembro. Os perfis reprodutivos apresentaram variação na atividade hormonal, durante a fase reprodutiva. Porém, comparando-se casais reprodutores com não-reprodutores, observa-se que os níveis de progestágenos não diferiram significativamente entre fêmeas que fizeram postura e aquelas que não fizeram. Entretanto, os níveis de andrógenos diferiram significativamente entre reprodutores e não-reprodutores. Diferenças hormonais foram observadas nas diferentes estações anuais, tanto nos machos, quanto nas fêmeas. Dados metereológicos de temperatura e insolação demostraram correlação negativa com os perfís anuais de progestágenos. A utilização da metodologia aqui mencionada demonstrou aplicabilidade, praticidade e segurança como ferramenta para o estudo da fisiologia e do manejo reprodutivos de Amazona vinacea. / During the last two decades, the techniques of analysis of fecal steroids have been applied for studying reproductive profiles in many species of captive wild and free living wild animals. The advantages of this methodology are widely understood. This study aimed to validate the anti- CL425 progestin and anti-R156/7 androgen enzyme immunoassay in fecal samples from parrot-breasted-purple (Amazon vinacea). This communication describes the annual endocrine profiles of urofecal sex steroids in Amazona vinacea with potential application for improving the species reproductive management. In total, 10 adult A. vinacea pairs kept in suspended aviaries in southern Brazil were studied. Excreta samples were collected twice weekly between May 2012 and May 2013. Gonadal activity was monitored by measurement of androgen and progestin metabolites in excreta of birds. Fresh excreta samples were collected always between 12:00 and 16:00 hs, and kept frozen until processing. The samples were dried at 57oC, hormones crushed and extracted using 80% methanol. The hormone dosage was performed at the Laboratory of Endocrinology, NUPECCE (Center for Research and Conservation of Deer, Jaboticabal, SP) using enzyme immunoassay with antibody to androgen and progestin. Feces from 5 A. vinacea pairs were used in two distinct periods for assay validation, within and outside the breeding season. Tests included Parallelism, Dose-Response, and Physiological Validation. It was concluded that the enzyme immunoassay for determining progestin and androgen profiles in urofecal samples from A. vinacea is accurate, precise and reliable. The annual profile of androgen in excreta of A. vinacea resulted the following monthly averages: January 25.9 ± 3.7 ng/g (n = 90); February 28.7 ± 3.2 ng/g (n = 60); March 31 7 ± 17.7 ng/g (n = 80); April 27.9 ± 5.5 ng/g (n = 90); May 43.3 ± 7.9 ng/g (n = 90); June 49.1 ± 15.9 ng/g (n = 80); July 36.5 ± 7.5 ng/g (n = 90); August 86.9 ± 46.2 ng/g (n = 90); September 42 125.7 ± 1 ng/g (n = 80); October 110.3 ± 63.2 ng/g (n = 90); November 74.2 ± 57.7 ng/g (n = 90) and December 43.3 ± 13 4 ng/g (n = 80). The monthly averages in the annual profile of progestin in excreta of A. vinacea were as follows: January 7.2 ± 0.9 ng/g (n = 90); February 9.26 ± 1.3 ng/g (n = 60); March 15 8 ± 2.9 ng/g (n = 80); April 14.15 ± 1.85 ng/g (n = 90); May 16.05 ± 3.16 ng/g (n = 90); June 11.85 ± 2.54 ng/g (n = 80); July 11.89 ± 2.17 ng/g (n = 90); August 13.52 ± 3.48 ng/g (n = 90); September 55 ± 45.7 ng/g (n = 80); October 15.44 ± 24.91 ng/g (n = 90); November 4.27 ± 6.42 ng/g (n = 90) and December 2.76 ± 0.42 ng/g (n = 80). Reproductive profiles showed variation in hormonal activity during the reproductive phase. When comparing breeding to non-breeding pairs, it was observed that the progestin levels did not differ significantly between laying and not-laying birds. However, the androgen levels differed significantly between breeding and non-breeding birds. In addition, hormonal differences were observed in both male and female birds in accordance with different annual seasons. Meteorological temperature and insolation data showed a negative correlation with the annual progestin profiles. Measurement of androgen and progestin metabolites in A. vinacea feces proved to be a practical and safe tool for studying reproductive physiology and management of the species.

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

Durante as últimas duas décadas, as técnicas de análise de esteroides fecais têm sido utilizadas para pesquisa em endocrinologia reprodutiva em diversas espécies animais silvestres de vida livre, ou cativeiro. O presente estudo propõe a validação da técnica do ensaio imunoenzimático antiprogestinas CL425 e antiandrógenos R156/7 fecais em papagaio-de-peito-roxo (Amazona vinacea). Objetiva-se descrever os perfis endócrinos anuais dos esteroides sexuais urofecais em Amazona vinacea com potencial aplicação para o aprimoramento do manejo reprodutivo da espécie. Foram utilizados 10 casais adultos da espécie Amazona vinacea mantidos em viveiros suspensos, em Lomba Grande, Rio Grande do Sul, Brasil. As excretas foram coletadas duas vezes por semana, entre maio de 2012 e maio de 2013. O monitoramento da atividade gonadal foi feito por mensuração de metabólitos de andrógenos nas excretas dos machos e progestágenos, nas de fêmeas. Foram coletadas amostras frescas de excretas, consistentemente, entre 12h00min e 16h00min e mantidas congeladas até o processamento. O perfil anual de andrógenos nas excretas de machos de A. vinacea apresentou médias mensais variáveis de 25,9 ng/g, em janeiro até 125,7 ng/g, em setembro. O perfil anual de progestinas nas excretas de fêmeas A. vinacea, as médias mensais variaram de 2,76 ng/g, em dezembro até 45,7 ng/g, em setembro. Os perfis reprodutivos apresentaram variação na atividade hormonal, durante a fase reprodutiva. Porém, comparando-se casais reprodutores com não-reprodutores, observa-se que os níveis de progestágenos não diferiram significativamente entre fêmeas que fizeram postura e aquelas que não fizeram. Entretanto, os níveis de andrógenos diferiram significativamente entre reprodutores e não-reprodutores. Diferenças hormonais foram observadas nas diferentes estações anuais, tanto nos machos, quanto nas fêmeas. Dados metereológicos de temperatura e insolação demostraram correlação negativa com os perfís anuais de progestágenos. A utilização da metodologia aqui mencionada demonstrou aplicabilidade, praticidade e segurança como ferramenta para o estudo da fisiologia e do manejo reprodutivos de Amazona vinacea. / During the last two decades, the techniques of analysis of fecal steroids have been applied for studying reproductive profiles in many species of captive wild and free living wild animals. The advantages of this methodology are widely understood. This study aimed to validate the anti- CL425 progestin and anti-R156/7 androgen enzyme immunoassay in fecal samples from parrot-breasted-purple (Amazon vinacea). This communication describes the annual endocrine profiles of urofecal sex steroids in Amazona vinacea with potential application for improving the species reproductive management. In total, 10 adult A. vinacea pairs kept in suspended aviaries in southern Brazil were studied. Excreta samples were collected twice weekly between May 2012 and May 2013. Gonadal activity was monitored by measurement of androgen and progestin metabolites in excreta of birds. Fresh excreta samples were collected always between 12:00 and 16:00 hs, and kept frozen until processing. The samples were dried at 57oC, hormones crushed and extracted using 80% methanol. The hormone dosage was performed at the Laboratory of Endocrinology, NUPECCE (Center for Research and Conservation of Deer, Jaboticabal, SP) using enzyme immunoassay with antibody to androgen and progestin. Feces from 5 A. vinacea pairs were used in two distinct periods for assay validation, within and outside the breeding season. Tests included Parallelism, Dose-Response, and Physiological Validation. It was concluded that the enzyme immunoassay for determining progestin and androgen profiles in urofecal samples from A. vinacea is accurate, precise and reliable. The annual profile of androgen in excreta of A. vinacea resulted the following monthly averages: January 25.9 ± 3.7 ng/g (n = 90); February 28.7 ± 3.2 ng/g (n = 60); March 31 7 ± 17.7 ng/g (n = 80); April 27.9 ± 5.5 ng/g (n = 90); May 43.3 ± 7.9 ng/g (n = 90); June 49.1 ± 15.9 ng/g (n = 80); July 36.5 ± 7.5 ng/g (n = 90); August 86.9 ± 46.2 ng/g (n = 90); September 42 125.7 ± 1 ng/g (n = 80); October 110.3 ± 63.2 ng/g (n = 90); November 74.2 ± 57.7 ng/g (n = 90) and December 43.3 ± 13 4 ng/g (n = 80). The monthly averages in the annual profile of progestin in excreta of A. vinacea were as follows: January 7.2 ± 0.9 ng/g (n = 90); February 9.26 ± 1.3 ng/g (n = 60); March 15 8 ± 2.9 ng/g (n = 80); April 14.15 ± 1.85 ng/g (n = 90); May 16.05 ± 3.16 ng/g (n = 90); June 11.85 ± 2.54 ng/g (n = 80); July 11.89 ± 2.17 ng/g (n = 90); August 13.52 ± 3.48 ng/g (n = 90); September 55 ± 45.7 ng/g (n = 80); October 15.44 ± 24.91 ng/g (n = 90); November 4.27 ± 6.42 ng/g (n = 90) and December 2.76 ± 0.42 ng/g (n = 80). Reproductive profiles showed variation in hormonal activity during the reproductive phase. When comparing breeding to non-breeding pairs, it was observed that the progestin levels did not differ significantly between laying and not-laying birds. However, the androgen levels differed significantly between breeding and non-breeding birds. In addition, hormonal differences were observed in both male and female birds in accordance with different annual seasons. Meteorological temperature and insolation data showed a negative correlation with the annual progestin profiles. Measurement of androgen and progestin metabolites in A. vinacea feces proved to be a practical and safe tool for studying reproductive physiology and management of the species.

Submitted by Mario BC (mario@bc.ufrpe.br) on 2016-08-03T12:58:52Z No. of bitstreams: 1 Felipe Rosendo Correia.pdf: 700962 bytes, checksum: ff53801d4695f2af9f4b25d3aae0ac55 (MD5) / Made available in DSpace on 2016-08-03T12:58:52Z (GMT). No. of bitstreams: 1 Felipe Rosendo Correia.pdf: 700962 bytes, checksum: ff53801d4695f2af9f4b25d3aae0ac55 (MD5) Previous issue date: 2016-02-22 / Currently there is a growing worldwide demand for milk and as a productive areas are increasingly limited, it is necessary to seek a higher efficiency to produce more smaller area. The use of technology is fundamental for development, artificial insemination at fixed time (IATF) is a biotechnology that increases the reproductive performance of the herd and when associated with a nutritional strategy makes the most productive activity. Supplementation with protected fat to ruminants has shown good results with increased reproductive rates and productive livestock. The objective of this experiment was to evaluate the endocrine and metabolic response of dairy cows of the breed Girolando by supplemental protected fat (Megalac-E®, QGN, RJ, Brazil) based on calcium salts with polyunsaturated fatty acids (PF) by determining the hormonal profile, energy, protein and mineral associating the increase in reproductive rates of animals that underwent artificial insemination at fixed time (IATF). Were synchronized 35 multiparous lactating cows divided into two groups, the control group (G1) with conventional farm diet and treated group (G2) with addition of 250g of Megalac-E® (QGN, RJ, Brazil) in the diet during the beginning of sync until the diagnosis of pregnancy. It assessed whether supplementation with protected fat caused changes in the endocrine and metabolic profile by associating with changes in the pregnant rate of treated animals. Variation between different variability factors, verifying that, for the groups factor, differences were observed for glucose, cholesterol, urea, creatinine, uric acid, globulin, phosphorus, progesterone and T4. As for the collections factor changes were observed for glucose, fructosamine, β-hydroxybutyrate, total protein, urea, creatinine, uric acid, globulin, calcium, phosphorus, triglyceride and T3. Regarding the interaction between the factors Groups x collections, only to glucose and β-hydroxybutyrate were registered significant variations. There was no variation in the concentration of any endpoint in the milk of cows control and receiving Megalac-E both empty as pregnant. The pregnancy rate was higher , no group supplemented with Megalac-E. Supplementation with protected fat (Megalac - E®) in the diet of cows Girolando submitted to FRAI promotes proper metabolic answer, running as a precursor paragraph Metabolic regulation, without causing nutritional and metabolic disorders , in addition to promote increase in pregnant rate and maintain a milk quality with good chemical characteristics and cellularity. / Há uma crescente demanda pelo leite a nível mundial e, como as áreas territoriais produtivas estão cada vez mais limitadas para este fim, torna-se necessário buscar uma maior eficiência para produzir mais em menor área. O emprego da tecnologia é fundamental, sendo a inseminação artificial em tempo fixo (IATF) uma biotecnologia responsável por aumenta a eficiência reprodutiva do rebanho e, que quando associada a uma estratégia nutricional, torna a atividade pecuária mais produtiva. A suplementação com gordura protegida para ruminantes tem demonstrado bons resultados, com aumento dos índices reprodutivos e produtivos dos rebanhos. O objetivo deste experimento foi avaliar a resposta endócrina, metabólica, composição de leite e taxa de prenhez em vacas leiteiras da raça Girolando submetidas à IATF e suplementadas com gordura protegida (Megalac-E®, QGN, RJ, Brasil) a base de sais de cálcio e ácidos graxos poli-insaturados (PF), determinar os perfis hormonal, energético, proteico e mineral associando ao aumento nos índices reprodutivos dos animais. Foram sincronizadas 35 vacas multíparas, em lactação, divididas em dois grupos: o grupo controle (G1), com dieta convencional da fazenda, e o grupo tratado (G2), com adição de 250g do Megalac-E® (QGN, RJ, Brasil) à dieta convencional durante o inicio da sincronização, até o diagnostico de gestação. Foi avaliado se a suplementação com gordura protegida causou alterações no perfil endócrino e metabólico, associando com alterações na taxa de prenhez dos animais tratados. No soro e plasma sanguíneo, houve variação entre os diferentes fatores de variabilidade, verificando-se que, para o fator Grupos, diferenças foram observadas para glicose, colesterol, ureia, creatinina, ácido úrico, globulina, fósforo, progesterona e T4. Quanto ao fator Coletas, foram observadas variações para glicose, frutosamina, β-Hidroxibutirato, proteína total, ureia, creatinina, ácido úrico, globulina, cálcio, fósforo, triglicerídeos e T3. Já em relação à interação entre os fatores Grupos x Coletas, apenas para glicose e β-Hidroxibutirato registraram-se variações significativas. Não houve variação na concentração de gordura, proteína, lactose, sólidos totais, sólidos não gordurosos e CCS do leite das vacas controle e as que receberam Megalac-E, tanto vazias quanto prenhas. A taxa de prenhez foi superior no grupo suplementado com o Megalac-E. Assim, considera-se que a suplementação com gordura protegida (Megalac-E®) na dieta de vacas Girolando submetidas à IATF promove adequada resposta metabólica e hormonal, por funcionar como precursor para a regulação metabólica, sem causar transtornos nutricionais e metabólicos, além de promover aumento na taxa de prenhez e de manter a qualidade do leite com boas características químicas e da celularidade.

Ginger belongs to the family of Zingiberaceae and has been utilized by mankind since ancient times to cure the common cold, headaches, nausea and stomach ache. Scientific studies have shown that ginger possess anti-microbial, anti-inflammatory, antipyretic, antioxidative, hypoglycemic, hepatoprotective and diuretic characteristics. Newly conducted studies have indicated that ginger also has antidiabetic effects. Ginger contains several substances and two of these are the enolic compounds gingerol and shogaol which may contribute to gingers antidiabetic effect. Diabetes type 2 is a condition where the pancreas can´t produce a sufficient amount of insulin, mainly due to insulin resistance. Increased blood glucose levels can eventually damage both large and small blood vessels which increases the risk of developing atherosclerosis. When the small blood vessels are damaged it can affect the blood supply to the eyes, kidneys and various parts of the nervous system. This could lead to reduced vision, kidney failure and decreased blood circulation. Firsthand treatment for type 2 diabetes is lifestyle changes which include dietary changes, increased exercise/activity and weight loss. When this isn’t enough oral medication, that stimulate the pancreas insulin production and increases the tissues sensitivity to insulin, is given. The purpose of this report is to answer the question: Can ginger be used as a complement to pharmaceutical treatment of diabetes type 2? This is a literature study where Pubmed and Google Scholar were used to search for scientific studies about gingers effect on patients with diabetes type 2. The result showed that ginger has a hypoglycemic effect in patients with type 2 diabetes. This effect seems to be dependent on dosage. Ginger seems to affect insulin resistance and insulin sensitivity in a positive way. The result also shows that ginger affect blood lipids, this result varied in the different studies, but conclusively it showed that it could decrease triglycerides and low density lipoprotein. It can occur a mild chronic inflammation in patients with type 2 diabetes. Ginger is known to be anti-inflammatory which can be considered an antidiabetic effect. The result in this study shows that some inflammatory markers decrease in patients with diabetes type 2. Despite different results all the studies in this report show that ginger has several antidiabetic effects. Ginger seems to have a positive effect on blood glucose levels, insulin resistance, blood lipids and inflammation. So to answer the question: Can ginger be used as a complement to pharmaceutical treatment of diabetes type 2? Yes, ginger seems to contribute to a antidiabetic effect and would be an good addition to the regular treatment of a patient with diabetes type 2.