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Defining the functional response of Mycobacterium sp. infected macrophages to structural variants of Interferon gamma (IFN-y)
Thesis (PhD)--Stellenbosch University, 2026.
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| Format: | Thesis |
| Language: | English |
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Stellenbosch : Stellenbosch University
2026
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| _version_ | 1867613738249486336 |
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| access_status_str | Open Access |
| author | Nyangwa, Soyama Sivatho |
| author2 | Kenyon, Colin P. |
| author_browse | Kenyon, Colin P. Nyangwa, Soyama Sivatho |
| author_facet | Kenyon, Colin P. Nyangwa, Soyama Sivatho |
| author_sort | Nyangwa, Soyama Sivatho |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (PhD)--Stellenbosch University, 2026. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/136039 |
| institution | Stellenbosch University (South Africa) |
| language | English |
| last_indexed | 2026-06-10T12:40:54.953Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2026 |
| publishDateRange | 2026 |
| publishDateSort | 2026 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/136039 Defining the functional response of Mycobacterium sp. infected macrophages to structural variants of Interferon gamma (IFN-y) Nyangwa, Soyama Sivatho Kenyon, Colin P. Walzl, Gerhard Kleynhans, Leanie Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Division of Molecular Biology and Human Genetics. Thesis (PhD)--Stellenbosch University, 2026. Nyangwa, S. S. 2026. Defining the functional response of Mycobacterium sp. infected macrophages to structural variants of Interferon gamma (IFN-y). Unpublished doctoral dissertation. Stellenbosch: Stellenbosch University [online]. Available: https://scholar.sun.ac.za/items/05c1604e-cb16-4f06-a8cf-9f4d3806ec44 Interferon-gamma (IFN-γ) is a key cytokine that orchestrates immune defence through its antiviral, antimicrobial, and antitumour activities. Despite its central role in immune regulation, its broader therapeutic application has been constrained by an incomplete understanding of how subtle structural variation influences its conformational organisation and stability. In biological systems, IFN-γ exists not as a single uniform molecular entity but as a structurally heterogeneous population arising from post-translational modification, proteolytic processing, and expression context. However, this heterogeneity is rarely considered in current diagnostic and therapeutic approaches, which typically treat IFN-γ as structurally invariant. This study aimed to address this gap by investigating how defined structural variation influences the conformational organisation of recombinant human IFN-γ. To this end, engineered IFN-γ variants representing C-terminal truncation and signal peptide retention were designed, cloned, and expressed in both Escherichia coli and HEK 293 cells. The resulting proteins were purified and analysed using circular dichroism (CD) and intrinsic fluorescence spectroscopy to probe secondary and tertiary structural organisation. This approach enabled direct comparison of structural behaviour between wild-type and variant proteins, as well as across different expression systems. CD analysis demonstrated that wild-type IFN-γ retained the characteristic negative bands at 208 nm and 222 nm, consistent with a predominantly α-helical structure. In contrast, truncated variants including 124A, 124B, and 133 exhibited reduced ellipticity at these wavelengths, indicating decreased helical content and altered secondary structural organisation. Intrinsic fluorescence spectroscopy further revealed reproducible shifts in emission maxima, consistent with changes in aromatic residue environments and perturbations in tertiary structure. Collectively, these findings demonstrate that truncation near the C-terminal region is associated with measurable and consistent alterations in IFN-γ structural integrity. Comparative expression analysis further revealed that expression system influences conformational organisation. While E. coli expression yielded higher protein quantities, IFN-γ produced in HEK 293 cells exhibited spectroscopic features more closely resembling those reported for native cytokine, consistent with the influence of eukaryotic folding machinery and post-translational context. These observations underscore the importance of expression-host selection when producing structurally representative recombinant cytokines. Overall, this study supports an ensemble-based view of human IFN-γ as a conformationally heterogeneous cytokine whose structural organisation is sensitive to sequence modification and expression environment. Beyond providing new structural insight into IFN-γ, the work establishes a transferable biophysical framework for interrogating structural heterogeneity in other cytokines and protein therapeutics. By defining the structural consequences of IFN-γ heterogeneity, this thesis lays a foundation for future functional and mechanistic investigations rather than asserting direct biological outcomes. Doctoral 2026-04-21T08:09:41Z 2026-04-21T08:09:41Z 2026-03 Thesis https://scholar.sun.ac.za/handle/10019.1/136039 en Stellenbosch University 265 pages application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Nyangwa, Soyama Sivatho Defining the functional response of Mycobacterium sp. infected macrophages to structural variants of Interferon gamma (IFN-y) |
| title | Defining the functional response of Mycobacterium sp. infected macrophages to structural variants of Interferon gamma (IFN-y) |
| title_full | Defining the functional response of Mycobacterium sp. infected macrophages to structural variants of Interferon gamma (IFN-y) |
| title_fullStr | Defining the functional response of Mycobacterium sp. infected macrophages to structural variants of Interferon gamma (IFN-y) |
| title_full_unstemmed | Defining the functional response of Mycobacterium sp. infected macrophages to structural variants of Interferon gamma (IFN-y) |
| title_short | Defining the functional response of Mycobacterium sp. infected macrophages to structural variants of Interferon gamma (IFN-y) |
| title_sort | defining the functional response of mycobacterium sp infected macrophages to structural variants of interferon gamma ifn y |
| url | https://scholar.sun.ac.za/handle/10019.1/136039 |
| work_keys_str_mv | AT nyangwasoyamasivatho definingthefunctionalresponseofmycobacteriumspinfectedmacrophagestostructuralvariantsofinterferongammaifny |