published_or_final_version / Zoology / Master / Master of Philosophy

published_or_final_version / Zoology / Master / Master of Philosophy

Cyclic AMP-dependent protein kinase (cAMP-PK) is an important mediator of hormone action. Its activity ratio is an accurate indicator of cellular activity under various experimental conditions including: (1) age and sex, (2) hormone administration and (3) temperature and photoperiod. Pituitary activity in unstimulated birds is not altered by age, but thyroid activity is much higher in old birds than in young animals. Thyrotropin releasing hormone (TRH) increases pituitary, thyroid and liver activity of prepubescent chickens, but has no effect on aged males and increases only thyroid and liver activities in aged females, suggesting a reduction in pituitary-thyroid function with advancing age. In prepubertal females, TRH increases pituitary and thyroid cAMP-PK activity, plasma T₃ and T₄ levels and liver T₄ monodeiodination. Thyroid activity reaches maximum activity before the pituitary, while plasma T₄ and liver T₄ monodeodinating activity reach their highest levels 20 minutes before plasma T₃. These findings suggest that fluctuations in liver T₄ 5' monodeiodinating activity might be responsible for the cyclic response of plasma T₃ and T₄. Castrated cockerels have larger pituitaries than untreated birds, but contain the same amount of DNA. Methimazole-fed cockerels have pituitaries significantly smaller than controls, while castrated cockerels fed methimazole have pituitaries the same size as untreated birds. Pituitary DNA is less than controls in both groups of methimazole-fed birds. These results are considered to be due to a change in the thyrotroph population, without an increase in total cell numbers, and may indicate a transformation of basophils. Pituitary cAMP-PK activity during cold stress substantiates this conclusion. Thyroid glands of castrated and untreated cockerels are smaller in size, histological appearance and DNA content; however, cAMP-PK activity is much greater in the castrated birds. Methimazole-fed cockerels have enlarged thyroid glands, elevated cAMP-PK activity, increased DNA and cellular hypertrophy; however, these effects may be mitigated by castration. Seven days after removal of testosterone supplements, photostimulated castrates have a higher thyroid cAMP-PK activity ratio than short day castrates; however, both groups are elevated above control, suggesting that long photoperiods enhance the stimulatory effects of castration on thyroid activity. Pituitary activity is elevated in long and short day birds seven days after removal of testosterone, but remains high only in short day castrates. Therefore, a reduction in the sensitivity of the hypothalamic-pituitary axis to testosterone may occur only in long day cockerels.

No description available.

Sexual selection and the maintenance of species diversity in Lake Malawi cichlid fishes are greatly dependent on optical communication, which is influenced by environmental, physiological and endocrinological factors. The diversity in spectral sensitivity of cichlids has been partially attributed to differences in opsin gene expression, with each species preferentially expressing a subset of seven possible genes. Hormones such as gonadotropin releasing hormone (GnRH) can mediate changes in gene expression and the presence of GnRH immunoreactive fibers and GnRH receptors throughout the retinal layers make it an excellent candidate for mediating changes in visual processes. Effects of exogenous GnRH administration on the visual system of zebra cichlids (Metriaclima zebra) via prolonged release cholesterol implants and intubation was investigated using electroretinogram (ERG) recordings, quantitative real-time RT-PCR and in situ hybridization. Three week and ten week sampling periods were used in the intubation study. No obvious differences in spectral sensitivity were evident when looking at a-wave, b-wave and d-wave components of the ERG waveform in any of the treatment groups. A multiple mechanism model was used to describe the cone mechanisms mediating spectral sensitivity and this analysis showed that the activity of cones was shaped by opponent and non opponent cone interactions based on subsets of five opsin genes previously described in cichlids (SWS1, SWS2b, RH2b, RH2aβ, and RH2aα). Although differences in the spectral sensitivity between control and GnRH-treated fish were not evident on a functional level, there were changes in the gonadosomatic index in the intubation group. Quantitative real-time RT-PCR (qRT-PCR) and in situ hybridization demonstrated that treatment with a synthetic GnRH3 analogue using the oral intubation delivery system resulted in statistically significant changes in opsin gene expression in both three week and ten week treatment groups, specifically the upregulation of RH2b and the downregulation of RH2a opsin genes. Moreover, in situ hybridization analysis showed that the pattern of labeling for the RH2a and RH2b riboprobes corroborated the changes in opsin gene expression found in the qRT-PCR data. In contrast, GnRH treatment using the cholesterol implant delivery system did not result in significant changes in spectral sensitivity or opsin gene expression. / Thesis (Master, Biology) -- Queen's University, 2011-01-05 22:57:11.308

This thesis is focused on the development, preclinical evaluation, and optimization of radiotracers for the detection of gastrin-releasing peptide receptor (GRPR)-expressing tumors. The work is divided into three distinct parts: (1) the development of bombesin (BN) antagonist (RM26)-based imaging radiotracers for the detection of GRPR-expressing tumors using different positron emission tomography (PET) and single photon emission computed tomography (SPECT) radionuclides (68Ga, 18F and 111In), (2) the establishment of a method to monitor the ligand-G protein-coupled receptor (GPCR) interaction in real time without requiring purification and stabilization of the receptors, and (3) the evaluation of radiopeptide structure-related factors (length of mini-PEG linker and composition of chelator for metal labeling) affecting the in vitro and in vivo characteristics of RM26-based tracers. We demonstrated the possibility of high-contrast in vivo imaging of GRPR-expressing xenografts despite the physiological expression of GRPR in abdominal organs. Fast radioactivity clearance from the blood and healthy organs, including receptor-positive organs, and long retention in the tumors resulted in high tumor-to-background ratios. A novel real-time assay for measuring the kinetics of the radiotracers targeting GPCR was evaluated. Living cells were used instead of purified receptors in this technology, bringing the developmental work one step closer to the true target environment (imaging in living systems). The comparative study of 68Ga-labeled NOTA-PEGn-RM26 with di-, tri-, tetra- and hexaethylene glycol chains demonstrated that the addition of only a few units of ethylene glycol to the spacer is insufficient to appreciably affect the biodistribution of the radiopeptide. Finally, a comparative study of 68Ga-labeled PEG2-RM26 analogs N-terminally conjugated to NOTA, NODAGA, DOTA or DOTAGA highlighted the influence of the chelator on the targeting properties of the radiopeptide. The main conclusion that can be drawn from this thesis is that 68Ga-NOTA-PEG2-RM26 has favorable biodistribution properties, such as rapid clearance from blood and tissues with physiological GRPR expression levels and long retention in GRPR-expressing tumors, and that this radiopeptide is potentially suitable for initial clinical investigation.

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.

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.

Proefschrift Universiteit van Amsterdam. / Met bibliogr., lit. opg. - Met samenvatting in het Nederlands.