published_or_final_version / Zoology / Master / Master 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.

Juvenile hormones (JHs) are key regulators of both metamorphosis and adult reproductive processes. The role of two key enzymes in the biosynthetic pathway of JH were examined: Farnesoic Acid O-Methyltransferase (FAMeT) and Juvenile Hormone Acid Methyltransferase (JHAMT). In crustaceans, FAMeT has been found to methylate farnesoic acid (FA), producing methyl farnesoate (MF) prior to epoxidation at the penultimate stage of JH biosynthesis. JHAMT was discovered more recently in the silkworm Bombyx mori and converts epoxidated FA (JHacids) to active JH through methylation using S-adenosyl-L-methionine (SAM). The aim of the proposed research is to examine the influence of a) decreasing the amount of FAMeT produced using an enhancer trapping P-element and b) increasing the levels of JHAMT and FAMeT in specific tissues using GAL4 overexpression techniques. Immunohistochemical analysis was used to confirm the presence of FAMeT in the CA of D. melanogaster ring glands. Analysis of MF, JHIII and JHB3 release in wild type and mutant stocks in the presence and absence of Drome AST (PISCF-type) suggest that Drosophila FAMeT has little if any effect on the sesquiterpenoid biosynthesis. Drome-AST appears to have a select effect on JHB3 biosynthesis and not MF or JHIII. Analysis of JHB3 release from larval and adult flies ubiquitously overexpressing JHAMT showed a significant increase when compared to wildtype (p<0.01 and p<0.0001 respectively). No significant difference was seen in JHB3 release in flies ubiquitously overexpressing FAMeT. A significant increase in hatching success was seen in flies overexpressing FAMeT in the larval ring gland and oocytes (p<0.05) whereas no significant decrease was seen in JHAMT-overexpressing flies during development. A significant extension of lifespan was also seen when FAMeT was overexpressed in the border and follicle cells of the oocyte (p<0.0001). The direct role of JHAMT in JHB3 synthesis has been demonstrated. The involvement of FAMeT and JHAMT in development and longevity may require other interacting proteins to elicit an effect, which is a limiting factor in overexpression experiments of the two enzymes. Additionally, this is the first example of AST action within D. melanogaster. / Thesis (Master, Biology) -- Queen's University, 2007-09-27 20:08:23.69

The gonadotropin-releasing hormone (GnRH) neurons of the hypothalamus are the principal regulators of reproductive function and are strongly modulated by estrogen (E₂). Several studies indicate that E₂ is able to influence GnRH neurons, both with "classical" long-term transcriptional effects, and with rapid non-transcriptional effects. One most interesting action of E₂ is that of modulating intracellular calcium concentration [Ca�⁺]I: this has been shown to happen in many different cell types, including embryonic models of GnRH neurons. The aim of this project was to evaluate if these rapid effects of E₂ on [Ca�⁺]I also happen at the level of adult GnRH neurons. In order to study the acute effects of E₂ on calcium dynamics, a novel transgenic mouse line was generated, that allows real-time measurement of [Ca�⁺]I selectively in GnRH neurons in an acute brain slice preparation. Using this mouse line, our group has previously shown that these cells show spontaneous activity in the form of Ca�⁺ transients. A first set of experiments was designed to define the effects of E₂ on spontaneous activity. E₂ was found to modulate [Ca�⁺]I in a activity-dependent manner: it increased the frequency of [Ca�⁺]I transients in about 50% of GnRH neurons with low spontaneous activity, whereas it decreased the frequency of the transients in more than 80% spontaneously active GnRH neurons. Different experiments were then performed in order to determine the molecular pathways that generates these opposite effects. The inhibitory effect was reproduced by the membrane-impermeable compound E2-6-BSA, indicating that it happens through a membrane receptor. The E₂ isomer l7α-estradiol was also able to reproduce the inhibitory effect of E₂, suggesting the involvement of some non-classical receptor. This is also confirmed by the presence of this effect in estrogen-receptor β (ER-β) knock-out mice, which exclude the involvement of this receptor. The stimulatory effect was found to be generated through a novel, indirect mechanism. It cannot be reproduced by E2-6-BSA nor by l7α-estradiol, and it is still present in the ER-β knock-out mice. The stimulation, though, can be reproduced in about 50% of cells with an ER-α selective agonist. As this receptor is not present in GnRH neurons, an indirect mechanism must be generating the stimulatory effect. Blockage of action potential mediated synaptic transmission with tetrodotoxin (TTX) did not block E₂ effects, but blockage of non-action potential mediated GABAergic transmission using the GABA[A] selective blocker gabazine completely abolished them. Our hypothesis is therefore that E₂ stimulates the generation of [Ca�⁺]I transients through estrogen-receptor a (ER-α) located in the terminals of GABAergic afferents. This modulation, in turn, is able to determine release of Ca�⁺ from IP₃-sensitive intracellular stores. To confirm this, we applied exogenous GABA to the neurons and found that it was able to initiate [Ca�⁺]I transients. Furthermore, removal of tonic GABAergic tone with gabazine was able to block spontaneous activity. To further analyse the effects of E₂, Ca�⁺ imaging experiments were performed together with cell-attached patch clamp electrophysiological recordings in order to correlate the electrical activity with the calcium activity. Simultaneous recordings revealed a strong correlation between [Ca�⁺]I transients and bursts of action currents in adult GnRH neurons. E₂ was able to increase the electrical activity of GnRH neurons with low spontaneous activity, and inhibit that of highly active ones. Application of GABA to GnRH neurons resulted in increased firing, accompanied by an increase in [Ca�⁺]I. These observations provide evidence for a complex mechanism of E₂ action on adult GnRH neurons, that may be important for the generation of the pulsatile release of this hormone.

No description available.

The gonadotropin-releasing hormone (GnRH) neurons of the hypothalamus are the principal regulators of reproductive function and are strongly modulated by estrogen (E₂). Several studies indicate that E₂ is able to influence GnRH neurons, both with "classical" long-term transcriptional effects, and with rapid non-transcriptional effects. One most interesting action of E₂ is that of modulating intracellular calcium concentration [Ca�⁺]I: this has been shown to happen in many different cell types, including embryonic models of GnRH neurons. The aim of this project was to evaluate if these rapid effects of E₂ on [Ca�⁺]I also happen at the level of adult GnRH neurons. In order to study the acute effects of E₂ on calcium dynamics, a novel transgenic mouse line was generated, that allows real-time measurement of [Ca�⁺]I selectively in GnRH neurons in an acute brain slice preparation. Using this mouse line, our group has previously shown that these cells show spontaneous activity in the form of Ca�⁺ transients. A first set of experiments was designed to define the effects of E₂ on spontaneous activity. E₂ was found to modulate [Ca�⁺]I in a activity-dependent manner: it increased the frequency of [Ca�⁺]I transients in about 50% of GnRH neurons with low spontaneous activity, whereas it decreased the frequency of the transients in more than 80% spontaneously active GnRH neurons. Different experiments were then performed in order to determine the molecular pathways that generates these opposite effects. The inhibitory effect was reproduced by the membrane-impermeable compound E2-6-BSA, indicating that it happens through a membrane receptor. The E₂ isomer l7α-estradiol was also able to reproduce the inhibitory effect of E₂, suggesting the involvement of some non-classical receptor. This is also confirmed by the presence of this effect in estrogen-receptor β (ER-β) knock-out mice, which exclude the involvement of this receptor. The stimulatory effect was found to be generated through a novel, indirect mechanism. It cannot be reproduced by E2-6-BSA nor by l7α-estradiol, and it is still present in the ER-β knock-out mice. The stimulation, though, can be reproduced in about 50% of cells with an ER-α selective agonist. As this receptor is not present in GnRH neurons, an indirect mechanism must be generating the stimulatory effect. Blockage of action potential mediated synaptic transmission with tetrodotoxin (TTX) did not block E₂ effects, but blockage of non-action potential mediated GABAergic transmission using the GABA[A] selective blocker gabazine completely abolished them. Our hypothesis is therefore that E₂ stimulates the generation of [Ca�⁺]I transients through estrogen-receptor a (ER-α) located in the terminals of GABAergic afferents. This modulation, in turn, is able to determine release of Ca�⁺ from IP₃-sensitive intracellular stores. To confirm this, we applied exogenous GABA to the neurons and found that it was able to initiate [Ca�⁺]I transients. Furthermore, removal of tonic GABAergic tone with gabazine was able to block spontaneous activity. To further analyse the effects of E₂, Ca�⁺ imaging experiments were performed together with cell-attached patch clamp electrophysiological recordings in order to correlate the electrical activity with the calcium activity. Simultaneous recordings revealed a strong correlation between [Ca�⁺]I transients and bursts of action currents in adult GnRH neurons. E₂ was able to increase the electrical activity of GnRH neurons with low spontaneous activity, and inhibit that of highly active ones. Application of GABA to GnRH neurons resulted in increased firing, accompanied by an increase in [Ca�⁺]I. These observations provide evidence for a complex mechanism of E₂ action on adult GnRH neurons, that may be important for the generation of the pulsatile release of this hormone.

Thesis (M.S.)--University of Missouri-Columbia, 2007. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on March 31, 2008) Vita. Includes bibliographical references.

Thesis(M.S.)--Auburn University, 2006. / Abstract. Vita. Includes bibliographic references.