Enteroendocrine hormones released from the intestine following food intake have several roles in the control of metabolism, some of which are exploited therapeutically for the treatment of type 2 diabetes. Within this thesis, focus has been on the receptors of the gut hormones glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-2 (GLP-2). In recent years there has been a surge of interest in the enteroendocrine hormones particularly due to the success of GLP-1 mimetics in the treatment of type 2 diabetes. GLP-1 is an incretin hormone, which enhances glucose induced insulin secretion by binding GLP-1 receptors (GLP1R) on pancreatic β-cells. Despite the therapeutic success, several extra-pancreatic clinical effects of GLP-1 remain unexplained. Here, a GLP1R monoclonal antagonistic antibody that can block GLP1R signalling in vivo has been developed and characterised, providing a new tool for the study of GLP1R physiology. GIP is the second incretin hormone, initially referred to as the 'ugly duckling' incretin hormone due to it's ineffectiveness in inducing insulin secretion in type 2 diabetic patients. Aside from the incretin actions, GIP is thought to be involved in the regulation of high-fat diet (HFD) induced obesity. A new transgenic mouse model expressing a fluorescent reporter under the control of the Gipr promoter has been used here to identify GIPR expressing cells. This model showed GIPR expression in the pancreas, adipose tissue, duodenum and nodose ganglia. Surprisingly GIPR expressing cells were found centrally, in areas of the hypothalamus involved in the regulation of food intake and energy expenditure. We consequently sought to investigate the function of GIPR expressing hypothalamic cells. GLP-2, unlike GLP-1 and GIP, is not an incretin hormone. Rather, GLP-2 has been implicated in the regulation of epithelial cell proliferation and apoptosis within the intestine. Therapeutically, an analogue of GLP-2 is used for the treatment of short bowel syndrome. A common missense mutation in the GLP-2 receptor (GLP2R), D470N, has been found to be associated with type 2 diabetes, and here we sought to understand the mechanism underlying this association. The D470N mutant has decreased β-arrestin recruitment, though the significance of this finding will need further research. Overall; the new monoclonal antagonistic GLP1R antibody will help to further understand GLP1R physiology, the new transgenic GIPR mouse model has contributed to the understanding of GIPR localisation, and cell based assays have identified functional implications of a polymorphism in the GLP2R associated with an increased risk of diabetes. It is hoped that further understanding of the physiology of these gut hormone receptors will be critical in the development of new therapeutics for diabetes and obesity.

Orientador: João Carlos Pinheiro Ferreira / Abstract: Thirty Nelore cows were submitted to estrus synchronization using a single administration of lecirelina, at 25 mg dosage and seven days later, D+ Cloprostenol at, 0.15 mg being both administered intra-muscularly. The first 16 cows ovulating were selected and daily evaluation was performed by rectal palpation and ultrasound of the female reproductive tract followed by blood sampling. Endometrial samples were collected by biopsy at days 0, 5, 9, 13 and 19 of the estrous cycle to detect the presence of estrogen and progesterone receptors by immunohistochemistry. The cows were distributed in 3 groups: control (without endometrial sampling, n=8), D0 (sampling initiated at the day of ovulation, n=4) and D13 (sampling initiated at day 13 of the estrous cycle, n=4). The mean inter-estrus interval was 23.80 l 2.70. The progesterone plasma mean concentrations ranged from 0.06 ng/mL to 4.53 ng/mL, decreasing throughout the cycle and reaching the lowest value close to ovulation. The pattern was characterized by increasing values during the luteal phase. The estradiol 17-b mean concentrations ranged from 6.22 pg/mL to 14.10 pg/mL and these values did not change throughout the estrous cycle. The results obtained by the rectal palpation revealed that the uterine tone and thickness were changing throughout the estrous cycle, being elevated during the period close to ovulation, and they were decreased during the progesterone phase. The ultrasound demonstrates that the amount of fluid content on the cranial vaginal compartment and in the uterine horn lumen was modified throughout the cycle. The largest amount of fluid was observed during the closest period to estrus and small fluid accumulation was observed during the progesterone phase. However, cows that were submitted to endometrial sampling presented more fluid during the progesterone phase... / Mestre

Thesis (Ph. D.)--University of Texas at Austin, 2006. / Vita. Includes bibliographical references.

published_or_final_version / Zoology / Doctoral / Doctor of Philosophy

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / abstract / toc / Zoology / Doctoral / Doctor of Philosophy

published_or_final_version / Zoology / Master / Master of Philosophy

published_or_final_version / Zoology / Master / Master of Philosophy

Several chemically-modified analogues of α-melanotropin (α-MSH, Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH₂) were prepared by solid-phase peptide synthesis, including [Nle⁴]-α-MSH, Ac-[Nle⁴]-α-MSH₄₋₁₃-NH₂, Ac-[Nle⁴]-α-MSH₁₋₆-NH₂, Ac-α-MSH₇₋₁₀-NH₂, Ac-α-MSH₁₁₋₁₃-NH₂, Ac-[Nle⁴]-α-MSH(,4-10)-NH₂, Ac-[Nle⁴, D-Phe⁷]-α-MSH₄₋₁₀-NH₂, [Nle⁴, D-Phe⁷]-α-MSH, Ac-α-MSH₄₋₁₀-NH₂, Ac-[Tyr⁴]-α-MSH₄₋₁₀-NH₂ and [half-Cys⁴, half-Cys¹⁰]-α-MSH. The synthetic strategy involved: (1) p-methylbenzhydrylamine resin as a solid support, (2) N,N'-dicyclohexylcarbodiimide as a coupling reagent, (3) acetylation of the N-terminus and HF cleavage and deprotection (except for Nⁱ-For-Trp) of the fully assembled peptide-resin and (4) alkaline hydrolysis to deformylate Nⁱ-For-Trp. In the preparation of [half-Cys⁴, half-Cys¹⁰]-α-MSH, oxidative-cyclization provided formation of an intramolecular disulfide bridge. A comparative biological analysis in vitro of these above structural, stereoisomeric and conformationally-restricted analogues of α-MSH on several different vertebrate pigment cell systems provided the following results: (1) The [Nle⁴, D-Phe⁷]-α-MSH effected high melanotropic potency (> 60 times relative to α-MSH), ultralong biological activity and unprecedented metabolic stability. (2) Utilizing [Nle⁴, D-Phe⁷]-α-MSH as a molecular probe, two melanotropic receptor types were demonstrated which were mechanistically different in terms of calcium dependency and apparent hormone-receptor complex reversibility. (3) The Ac-[Nle⁴, D-Phe⁷]-α-MSH₄₋₁₀-NH₂ was a highly potent active site (Met-Glu-His-Phe-Arg-Trp-Gly) analogue of α-MSH (ranging from 0.2- to 10-times relative to α-MSH) without the ultralong melanotropic activity possessed by the parent stereostructural tridecapeptide. (4) The [half-Cys4, half-Cys10]-α-MSH exhibited superpotency on frog (Rana pipiens) melanophores (≥ 10,000 times relative to α-MSH), and provided experimental evidence that a pseudocyclic conformation of the native hormone containing a β-turn structural requirement at His-Phe-Arg-Trp might be related to its biological activity at the pigment cell receptor. The [Nle⁴, D-Phe⁷]-α-MSH may be suitable for use as a radio-labeled tracer or drug-delivery agent for the localization or treatment of human melanoma in vivo.

The present investigation was designed to further our understanding of the interaction of pituitary (luteinizing hormone, LH) and placental (chorionic gonadotropin, CG) gonadotropins with the primate corpus luteum. Studies were performed (1) to characterize the LH/CG receptor population in the