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A Dissertation submitted to the Faculty of Health Science, University of the Witwatersrand, in fulfillment of the requirements for the degree of Master of Science. Johannesburg, 2017 / Gonadotropin-releasing hormone (GnRH) binds to GnRH receptors (GnRHR) in the pituitary and stimulates release of gonadotropins, which control reproduction. It has been proposed that the congenital Glu2.53(90)Lys GnRHR mutation causes infertility by disrupting a salt-bridge important for GnRHR protein expression. To investigate its role in GnRHR function, Glu2.53(90) was mutated to residues that mimic or remove its side-chain properties. Mutant receptors were assessed for inositol phosphate signaling and radioligand binding. Receptors with small or negatively-charged substitutions for Glu2.53(90) exhibited no measurable function. Stabilizing receptor expression by appending a carboxy-terminal tail recovered function of the Glu2.53(90)Lys and Glu2.53(90)Ala GnRHRs, but not the conservative Glu2.53(90)Asp mutant. Receptors with uncharged (Gln) or hydrophobic (Leu, Phe) substitutions that cannot form salt-bridges with Lys3.32(121) were fully functional. Although the positively-charged Arg substitution decreased binding affinity, it preserved GnRHR function, confirming that interaction with the positively-charged Lys3.32(121) is not required. Comparing the GnRHR with structurally-related G protein-coupled receptors revealed that the equivalent residue of rhodopsin, Met2.53(86), interacts with Trp6.48(265). Mutating Trp6.48(280) of the GnRHR to Ala and Arg disrupted GnRH-stimulated function, confirming a role in expression. The Trp6.48(280)Arg GnRHR with an appended carboxy-terminal tail had decreased GnRH binding affinity. The preserved function of mutant receptors with large hydrophobic or positively-charged amino acid substitutions suggests that the size of the Glu2.53(90) is important for stabilizing GnRHR structure. Decreased affinity of mutant receptors with larger (Arg) substitutions for Glu2.53(90) and Trp6.48(280) suggest that both residues make conserved intramolecular interactions that stabilize receptor protein expression and configure the extracellular GnRHR structure. (250 words) / MT2017

Doctor of Philosophy (PhD) / Reproductive physiology plays a vital role in population growth and vitality. Baseline data on reproductive physiology and a comprehensive knowledge of breeding biology are essential to conservation management. Great whales have been hunted from the 16th century to the present day. Although many populations are increasing there are populations with low or declining reproductive rates. In 2001 it was recommended to the International Whaling Commission that new techniques be developed to assess the internal physiology of great whales. This study, based on this recommendation, aims to develop analytical methods to assess reproductive hormones in cetacean blow samples and determine the feasibility of its use with free-swimming great whales. A method for the assessment of steroid hormone concentrations using liquid chromatography-mass spectrometry (LC-MS) was developed and validated. These methods were then used to determine testosterone and progesterone concentrations in saliva and blow of bottlenose dolphins. The stability of testosterone and progesterone was found to be a major issue. Without inhibitors, hormone concentrations increased by up to 65% over three hours at 21oC. Storing samples at low temperatures (-20oC or -80oC) slowed but did not cease the rate of change. The addition of inhibitors, manganese chloride and amoxycillin potassium/clavulanate, improved the stability of testosterone and progesterone. It is proposed that when using dolphin saliva and blow samples to measure reproductive hormones the samples are extracted as soon as possible after collection to prevent degradation. This study highlighted the need to address steroid hormone stability prior to any longterm biological program, to ensure that changes seen in hormone concentration are due to biological activity rather than storage. A technique to collect blow samples from free-swimming great whales was developed. This technique, in conjunction with the specially developed LC-MS methods allowed for the determination of testosterone and progesterone concentrations in humpback whale blow. The techniques developed in this study to determine reproductive hormones in cetacean saliva and blow have applications for both captive and wild population studies. In captive institutions, saliva and/or blow can be used to monitor reproductive cycling in both females and males. As it is noninvasive it can be used on a daily basis with minimal stress to the animals. The use of blow sampling has the capacity to improve our understanding of reproductive cycling in great whales as it can be used to sample animals in both the breeding and feeding areas. This technique may allow us to now examine whether reproductive dysfunction is playing a role in the slow recovery of critically endangered species such as the North Atlantic right whale.

Thesis (M. D. S.)--University of Hong Kong, 2004. / Title proper from title frame. Also available in printed format.

The first part of this research involves the development of a gold-nanoparticle based sandwich type immunosensor to identify trace amounts of human chorionic gonadotropin hormone based on the direct electrochemical detection of Au nanoparticles. The second part of this research is to design a biosensor that can be easily handled, has higher specificity, sensitivity, low-cost, and rapid response and has a better detection of growth hormone (GH). Current bioanalytical techniques have reported the difficulty to detect GH doping. This research aims to address the issue of measuring GH in small volumes, which has been challenging the limits of analytical detection systems. The electrochemical measurements utilize the redox activity of ferro/ferricyanide in cyclic voltammetry and impedance spectroscopy. The detection limit 10 pg/mL was observed for GH in 20 μL sample volume, which indicated that this versatile platform can be easily adapted for decentralized electrochemical immunosensing of clinically important proteins.

The first part of this research involves the development of a gold-nanoparticle based sandwich type immunosensor to identify trace amounts of human chorionic gonadotropin hormone based on the direct electrochemical detection of Au nanoparticles. The second part of this research is to design a biosensor that can be easily handled, has higher specificity, sensitivity, low-cost, and rapid response and has a better detection of growth hormone (GH). Current bioanalytical techniques have reported the difficulty to detect GH doping. This research aims to address the issue of measuring GH in small volumes, which has been challenging the limits of analytical detection systems. The electrochemical measurements utilize the redox activity of ferro/ferricyanide in cyclic voltammetry and impedance spectroscopy. The detection limit 10 pg/mL was observed for GH in 20 μL sample volume, which indicated that this versatile platform can be easily adapted for decentralized electrochemical immunosensing of clinically important proteins.

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published_or_final_version / Medicine / Doctoral / Doctor of Philosophy