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University of Pretoria

1. Fourie, Christle. Effects of clomiphene citrate on the expression of kisspeptin dynorphin A and neurokinin B in female Sprague-Dawley rats.

Degree: MSc, Physiology, 2016, University of Pretoria

Clomiphene citrate (CC) is the leading treatment for women with anovulatory infertility. The precise mechanism of action of the drug on the hypothalamic-pituitary-gonadal (HPG) axis has yet to be determined. Neurons expressing kisspeptin, neurokinin B (NKB) and dynorphin A (Dyn), collectively called KNDy neurons, in the arcuate nucleus (ARC) of the hypothalamus have been shown to play an integral role in the estradiol (E2) feedback pathways of the reproductive system in mammals. KNDy neurons are found in the ARC and the anteroventral periventricular nucleus (AVPV) in humans but have been predominantly reported to not express NKB and Dyn in rodents. The axons of these neurons project to the medial eminence (ME) in the region where the gonadotropin-releasing hormone (GnRH) terminals and fibres are located. It was hypothesised that CC upregulates the gene expression of kisspeptin and neurokinin B while down-regulating the gene expression of dynorphin A which results in a leutenizing hormone surge and an increase in oestradiol which causes ovulation. This was a randomized experiment which included 18 female Sprague-Dawley rats in which the aim was to analyse the expression of kisspeptin, NKB and Dyn in the ARC and the AVPV as well as blood plasma levels of oestradiol and leutinizing hormone (LH) in female rats after CC administration. Six of the rats constituted the control group that received a vehicle solution. The second group of 6 rats received the intervention in the form of CC and the third group of six rats received CC as well as p234-penetratin, a kisspeptin antagonist (KpA). The mRNA expression of the KNDy genes were analysed using real-time quantitative polymerase chain reaction (qPCR) and the plasma levels of E2 and LH were analysed by enzyme-linked immunosorbant assays (ELISA). ELISA results show that the E2 concentration in the group that received CC plus KpA was found to be marginally lower than that of the control group but there was no significant difference between the E2 concentrations of the control group and the group that received only CC. The LH concentration in the group that received CC plus KpA was significantly higher than both other groups but again, there was no significant difference between the LH concentration control group and the group that only received CC. The qPCR showed that in the AVPV, the kisspeptin expression of the CC group and the CC plus KpA groups are marginally higher than that of the control group. Conversely, the Dyn expression of the CC group and the CC plus KpA groups are marginally lower than that of the control group in the AVPV. There were no significant differences in NKB expression across the three groups. In the ARC, there were no significant differences in kisspeptin or Dyn expression across the groups. The NKB expression of the CC group was marginally lower than that of the control and there was no significant difference between the CC plus KpA group and the control group. In summary, CC appears to have a marginal effect on the kisspeptin and Dyn mRNA via the… Advisors/Committee Members: Millar, Robert Peter (advisor), Hlophe, Yvette (coadvisor), (Craig) (coadvisor).

Subjects/Keywords: UCTD; Clomiphene citrate; Ovulation induction; p234-penetratin; Kisspeptin

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APA (6th Edition):

Fourie, C. (2016). Effects of clomiphene citrate on the expression of kisspeptin dynorphin A and neurokinin B in female Sprague-Dawley rats. (Masters Thesis). University of Pretoria. Retrieved from http://hdl.handle.net/2263/61684

Chicago Manual of Style (16th Edition):

Fourie, Christle. “Effects of clomiphene citrate on the expression of kisspeptin dynorphin A and neurokinin B in female Sprague-Dawley rats.” 2016. Masters Thesis, University of Pretoria. Accessed March 09, 2021. http://hdl.handle.net/2263/61684.

MLA Handbook (7th Edition):

Fourie, Christle. “Effects of clomiphene citrate on the expression of kisspeptin dynorphin A and neurokinin B in female Sprague-Dawley rats.” 2016. Web. 09 Mar 2021.

Vancouver:

Fourie C. Effects of clomiphene citrate on the expression of kisspeptin dynorphin A and neurokinin B in female Sprague-Dawley rats. [Internet] [Masters thesis]. University of Pretoria; 2016. [cited 2021 Mar 09]. Available from: http://hdl.handle.net/2263/61684.

Council of Science Editors:

Fourie C. Effects of clomiphene citrate on the expression of kisspeptin dynorphin A and neurokinin B in female Sprague-Dawley rats. [Masters Thesis]. University of Pretoria; 2016. Available from: http://hdl.handle.net/2263/61684


Universiteit Utrecht

2. Walen, M. The efficacy of kisspeptin antagonists p234 and p271 in blocking the response to KP-10 in vivo in the bitch and in vitro using flow-cytometry.

Degree: 2014, Universiteit Utrecht

BACKGROUND: The neuropeptide kisspeptin and its receptor GPR54 play a crucial role in the reproductive function of mammals. Finding an efficient kisspeptin antagonist could be the first step towards a new method for non-surgical contraception in the dog. The aims of this study were to test kisspeptin antagonists 271 and 234 in vitro using flow-cytometry and to test p271 in vivo during different stages of the estrous cycle of the bitch. METHODS: In vitro: Chem-1 cells expressing the human GPR54 receptor were labeled with Fluo-3 AM, a fluorescence indicator for intracellular calcium, and examined by flow-cytometry. A concentration response study of hKP-10 and cKP-10 was designed to test if flow-cytometry is a reliable method for measuring intracellular calcium (as a measure for receptor activation) and to determine the optimal hKP-10 concentration. This data were then used to test the antagonists p234 and p271 in vitro. In vivo: P271 was tested during different stages of the estrous cycle of the bitch. In this study only the results in the follicular phase are discussed. Female dogs received 50 µg/kg/hour p271 by iv infusion for three hour. Two hours after the start of the infusion 0.5 µg/kg cKP-10 was iv administered. Controls received one iv injection of 0.5 µg/kg cKP-10. Blood samples before, during and after the peptide administration were collected for the determination of plasma [LH]. RESULTS: Both human and canine KP-10 gave a typical sigmoidal concentration-response curve in vitro. We found no antagonistic or agonist effect of peptide 234 and 271 in vitro. Furthermore, p271 was not able to reduce basal plasma LH concentrations and the LH response to cKP-10 in vivo in the bitch during follicular phase. CONCLUSION: P271 is probably not a good kisspeptin antagonist for the dog. Flow-cytometric calcium flux assays seem like a reliable method to study kisspeptin receptor activation in vitro. Advisors/Committee Members: Albers-Wolthers, C.H.J..

Subjects/Keywords: Kisspeptin; KP-10; GPR54; antagonist; p234; p271; in vitro; flow-cytometry; in vivo; bitch

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6th Edition):

Walen, M. (2014). The efficacy of kisspeptin antagonists p234 and p271 in blocking the response to KP-10 in vivo in the bitch and in vitro using flow-cytometry. (Masters Thesis). Universiteit Utrecht. Retrieved from http://dspace.library.uu.nl:8080/handle/1874/294313

Chicago Manual of Style (16th Edition):

Walen, M. “The efficacy of kisspeptin antagonists p234 and p271 in blocking the response to KP-10 in vivo in the bitch and in vitro using flow-cytometry.” 2014. Masters Thesis, Universiteit Utrecht. Accessed March 09, 2021. http://dspace.library.uu.nl:8080/handle/1874/294313.

MLA Handbook (7th Edition):

Walen, M. “The efficacy of kisspeptin antagonists p234 and p271 in blocking the response to KP-10 in vivo in the bitch and in vitro using flow-cytometry.” 2014. Web. 09 Mar 2021.

Vancouver:

Walen M. The efficacy of kisspeptin antagonists p234 and p271 in blocking the response to KP-10 in vivo in the bitch and in vitro using flow-cytometry. [Internet] [Masters thesis]. Universiteit Utrecht; 2014. [cited 2021 Mar 09]. Available from: http://dspace.library.uu.nl:8080/handle/1874/294313.

Council of Science Editors:

Walen M. The efficacy of kisspeptin antagonists p234 and p271 in blocking the response to KP-10 in vivo in the bitch and in vitro using flow-cytometry. [Masters Thesis]. Universiteit Utrecht; 2014. Available from: http://dspace.library.uu.nl:8080/handle/1874/294313

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