Browsing by Subject "Molecular Cell Biology"

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    Development of microalgae as a biopharming platform
    (2019) Els, Johann Hendrik; Rybicki, Edward; Hitzeroth Inga; Harrison, Sue
    Microalgae may be a powerful biopharmaceutical production platform that is still in its infancy of development. The research done in this project tested the feasibility of creating algal cell packs, a novel immobilised microalgae transient production platform for the expression of recombinant protein. First it had to be established whether the available plant expression vectors could be used for the transfer of genetic material into packed microalgae. The method showed successful transfer of the neomycin phosphotransferase II resistance gene (nptII). Further experiments analysed the plant expression vectors pTRAc and pRIC3.0 for expression of enhanced green fluorescent protein (EGFP) in Scenedesmus spp. by western blotting. Possible replication of the plant geminivirus-derived pRIC3.0 was then confirmed by comparing to replication in Nicotiana benthamiana by quantitative polymerase chain reaction (qPCR). Western blot results indicated EGFP expression in N. benthamiana but not in Scenedesmus. By using PCR the presence of EGFP DNA in Scenedesmus was detected but qPCR showed no increase of the pRIC3.0 replicon. Despite no detection via antibodies of EGFP in Scenedesmus, green fluorescence was observed. These initial results showed promise and points to a system that requires optimisation for increased transfection rates and protein expression. Following on from the initial work, the project set out to determine the feasibility of expressing a recombinant anti-Ebola viral inhibitor protein in three different plant based platforms namely N. benthamiana, a microalgal genus, Desmodesmus and a BY2 tobacco plant-cell culture. Protein expression was compared between the Desmodesmus algal cellpack, N. benthamiana plant expression system and BY-2 plant cell packs by western blotting. Four designs of the viral inhibitor fused to the maize ƴ-zein protein body inducing protein, ZERA, were expressed in trace quantities. Transient expression was more prominent in the algal cell packs than in N. benthamiana and BY-2 cells. The algal cell pack system may potentially be a powerful tool to test recombinant protein expression in a range of microalgal hosts via Agrobacterium-mediated genetic transfection. The future development of recombinant protein expression platforms could be enhanced by rapid testing of protein production in different species. Refinement needs to be done on the algal cell pack to increase transfection efficiency and expression in microalgae to produce commercially viable quantities of heterologous protein.
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    The regulation of the luteinizing hormone (LH) and the follicle stimulating hormone (FSH) by glucocorticoids and progestins
    (2025) Capitaine, Carole-Keza; Hapgood, Janet; Bick, Alexis
    Estradiol (E2) plays a crucial role in female reproduction and in the defense against HIV-1 in the female genital tract (FGT). Medroxyprogesterone acetate (MPA) intramuscular (DMPA-IM) (Depo-Provera) and norethisterone (NET) enanthate (NET-EN) are injectable progestin only contraceptives. DMPA-IM is commonly used in sub-Saharan Africa, while NET-EN is commonly used in South Africa. Controversial observational studies have demonstrated a heightened susceptibility to HIV-1 acquisition associated with the use of DMPA-IM but not NET-EN. The recent Women's Health, Injectable Contraception and HIV (WHICH) clinical trial compared E2 levels in equal numbers of women randomized to DMPA-IM (n=262) and NET-EN (n=259) found that both DMPA-IM and NET-EN use caused hypoestrogenism. Considering the widespread use of DMPA-IM and NET-EN among South African women, who are disproportionately affected by HIV-1, it is important to explore the mechanisms through which these contraceptives induce hypoestrogenism. These could potentially involve luteinizing hormone (LH), follicle-stimulating hormone (FSH), and gonadotropin-releasing hormone (GnRH), all hormones involved in the hypothalamic-pituitary-ovarian (HPO) axis and key regulators of E2 synthesis and release. This thesis analyzed the serum concentrations of E2, LH, FSH, and GnRH in women randomized to DMPA-IM (n = 100) or NET-EN (n = 93) in a subpopulation of the WHICH cohort. This thesis also involved a translational approach, comparing clinical data with mechanistic data from an in vitro model. Towards understanding the mechanisms whereby MPA and NET may regulate gonadotropin levels, it was hypothesized that these progestins exert direct actions on pituitary gonadotropes. It was further hypothesized that while both progestins are progestogenic and could act via the progesterone receptor (PR), MPA would most likely also exert glucocorticoid like actions via the glucocorticoid receptor (GR), unlike NET. These hypotheses were tested in the LβT2 mature mouse pituitary gonadotrope cell line. This in vitro model allowed the effects of MPA and NET on the gonadotropins at a transcriptional, post-transcriptional and secretion level to be tested. This study also investigated the effects of MPA, NET, dexamethasone (DEX, a GR agonist) and progesterone (P4, a PR agonist), in the absence and presence of GnRH, on LH and FSH regulation in the LβT2 cells. 3 Clinical results showed that E2 levels were similarly suppressed to postmenopausal levels by both contraceptives in the subpopulation of women. No effects were detected for either contraceptive on GnRH levels. LH levels decreased in both contraceptive groups, whereas FSH levels decreased in the NET-EN group and increased in the DMPA-IM group. The results in LβT2 cells showed that MPA and DEX both suppressed the GnRH-induced promoter-reporter activity of the beta subunit of LH (LHβ), with a non-significant decrease in GnRH-induced LH protein secretion. MPA and DEX increased the promoter-reporter activity and mRNA level of the beta subunit of FSH (FSHβ), while NET and P4 had no detectable change on FSH beta expression. Additionally, both MPA and DEX enhanced basal and GnRH-induced FSHβ promoter activity and mRNA levels. Taken together with GR antagonist experiments, the effects of MPA and DEX on FSHβ gene expression are likely mediated by the GR. Attempts to measure secreted FSH protein were unsuccessful. In summary, the combined clinical and in vitro data suggest that the hypoestrogenic effect caused by DMPA-IM and NET-EN in women is likely through direct actions of MPA and NET on pituitary gonadotropes to decrease LH levels. The decrease in LH levels in DMPA-IM users occurs most likely both at the level of gene transcription, as well as at the level of protein secretion, while this does not appear to be the case for NET-EN users. The increase in FSH levels in DMPA-IM users is likely due to direct effects by MPA on pituitary gonadotropes to increase FSHβ transcription and mRNA levels. This increase in FSH levels is likely due to the GR-mediated transcriptional regulation of FSHβ by MPA and not NET. The in vitro data do not explain the decrease in FSH levels detected in NET-EN users. However, there may be other unexplored mechanisms used by MPA and NET at the level of FSH secretion. The data also do not exclude that DMPA-IM and NET-EN may have other mechanisms that act at the ovarian level or in the hypothalamus or above the hypothalamus, to affect GnRH pulsatility. This study gives insight into previously unknown mechanisms involved in the regulation of gonadotropins by MPA and NET. This research also enhances our understanding of the potential mechanisms behind the hypoestrogenic effects caused by progestin-only injectable contraceptives.