Studies of mitophagy in mouse and fish models using antibodies and PCR

Simple item page
dc.contributor.advisorBellstedt, Dirk
dc.contributor.advisorLoos, Ben
dc.contributor.authorLeukes, Kay-Lynn Amber-Marie
dc.contributor.otherStellenbosch University. Faculty of Science. Dept. of Biochemistry.en_ZA
dc.date.accessioned2023-03-06T18:51:53Z
dc.date.accessioned2023-05-18T07:15:14Z
dc.date.available2023-03-06T18:51:53Z
dc.date.available2023-05-18T07:15:14Z
dc.date.issued2023-03
dc.descriptionThesis (MSc)--Stellenbosch University, 2023. en_ZA
dc.description.abstractENGLISH ABSTRACT: Parkinson’s Disease (PD) is a neurodegenerative progressive movement disorder that affects aging populations. Characterized by various motor and non-motor symptoms, it affects the daily lives of approximately 7-10 million people globally. It has significant socio-economic and mental effects on patients, and although various treatment and management methods exist and are available, PD is incurable, emphasising a strong need for further and extensive investigation into the underlying molecular pathways that cause it. Mitophagy is a process crucial for the regulation of degradation of dysfunctional mitochondria through autophagy. It upholds cellular homeostasis; however, its dysfunction has been linked to the development of PD and other neurodegenerative diseases. Various model organisms exist for the study of aging, Nothobranchius fish being one of the more recently emerging models due to their short lifespan in addition to the fact that these fish begin to display Parkinson’s-like Disease symptoms as they age. Numerous genes have been identified in these fish that have been linked to the development of these symptoms. This project therefore aimed to produce antibodies against two well-known proteins that have previously been used as molecular markers to study autophagy and mitophagy – LC3 and p62. The production of these antibodies allows for possible future studies of mitophagy and its role in PD- development. With aging being a major risk factor for the onset of neurodegenerative diseases, this project also aimed to investigate mitochondrial DNA (mtDNA) degradation in two aging groups of Nothobranchius species by DNA isolation and PCR analysis to observe mtDNA degradation with age. In this study, recombinant His6-LC3 and p62-KLH conjugates were used to immunize rabbits. Antigen specific enzyme-linked immunosorbent assays were used to confirm the production of anti-LC3 and anti-p62 antibodies. The characterization of the anti-LC3 antibodies was accomplished by means of western blotting and immunofluorescence and they were found to recognize the unlipidated, or unactivated form of LC3, LC3-I and not the lipidated form, LC3-II, which limits their usage in mitophagy activation studies. In spite of this, through affinity chromatography, and MagReSyn magnetic bead purification with these anti-LC3 antibodies, native LC3 from mitophagy activated mouse fibroblast (MEF) cells lines could be isolated for future potential antibody production against activated LC3-II. Characterization of anti-p62 antibodies was accomplished by means of western blotting and immunofluorescence, which yielded positive results. The antibodies raised against p62 possessed the same specificity as commercially available anti-p62 antibodies and can therefore be used as valuable tools in future studies of mitophagy in MEF cells. Due to the fact that these antibodies, based on the similarity of epitopes, were specific for p62 Nothobranchius epitopes, these antibodies may be of particular value for immunofluorescence studies of Nothobranchius mitophagy in future. The investigation into mtDNA degradation in Nothobranchius fish was attempted by PCR analyses with a variety of primer pairs of aging Nothobranchius korthausae and Nothobranchius guentheri in comparison with appropriate control samples. Mitochondrial degradation based on a reduction in amplification of large fragments of mtDNA from aging fish was not observed and calls for further and more in-depth investigation.en_ZA
dc.description.abstractAFRIKAANSE OPSOMMING: Parkinson se siekte (PS) is ‘n neurodegeneratiewe progressiewe bewegingsversteuring wat verouderende bevolkings beïnvloed. Gekenmerk deur verskeie motoriese en nie-motoriese simptome, beïnvloed hierdie siekte die daaglikse lewens van ongeveer 7-10 miljoen mense wêreldwyd. Dit het beduidende sosio-ekonomiese en geestelike versteuring effekte op pasiënte, en hoewel verskeie behandelings- en bestuursmetodes bestaan en beskikbaar is, is PS ongeneeslik, en beklemtoon ‘n sterk behoefte aan verdere en uitgebreide ondersoek na die onderliggende molekulêre weë wat dit veroorsaak. Mitofagie is ‘n proses wat absoluut noodsaaklik is vir die regulering van agteruitgang van disfunksionele mitochondria. Dit handhaaf sellulêre homeostase; en die disfunksie van hierdie proses is gekoppel aan die ontwikkeling van PS en ander neurodegeneratiewe siektes. Verskeie modelorganismes bestaan vir die studie van veroudering, Nothobranchius-visse is een van die meer onlangs opkomende modelle vanweë hul kort lewensduur, benewens die feit dat hierdie visse Parkinson-agtige siektesimptome begin vertoon namate hulle ouer word. Talle gene is in hierdie visse geïdentifiseer wat verband hou met die ontwikkeling van hierdie simptome. Hierdie projek het dus ten doel om teenliggame te produseer teen twee bekende proteïene wat voorheen as molekulêre merkers gebruik is om outofagie en mitofagy- LC3 en p62, te bestudeer. Die produksie van hierdie teenliggame maak voorsiening vir moontlike toekomstige studies van mitofagie en die rol daarvan in PS-ontwikkeling. Aangesien veroudering ‘n belangrike risikofaktor vir die aanvang van neurodegeneratiewe siektes is, het hierdie projek ook ten doel gehad om mitochondriale DNA (mtDNA) agteruitgang in twee verouderingsgroepe Nothobranchius-spesies deur DNA-isolasie en PKR-analise te ondersoek om die mtDNA-agteruitgang met ouderdom waar te neem. In hierdie studie, was rekombinante His6-LC3 en p62-KLH gebruik om konyne te immuniseer om ‘n immuunrespons te ontlok. Antigeen-spesifieke enzyme-linked immunosorbent assays (ELISA) was gebruik om die produksie van anti-LC3- en anti-p62-teenliggame te bepaal. Die karakterisering die anti-LC3 was gedoen met western klad en immunofluoressensie, wat gevind het die ongeaktiveerde vorm van LC3 LC3-I, en nie die geaktiveerde vorm, LC3-II, word herken. Dit het gelei tot sekere perke vir hul gebruik in studies van mitofagie. Ten spyte hiervan, kon inheemse LC3 vanuit mitofagie-geaktiveerde muis embrioniese fibroblastiese (MEF) sellyne deur middel van affiniteitschromatografie en MagReSyn magnetiese kraalsuiwering met hierdie teenliggame geïsoleer word vir toekomstige produksie van teenliggame teen geaktiveerde LC3 (LC3-II). Die karakterisering van anti-p62 teenliggame was bewerkstellig deur middel van Westersn klad en immunofluoressensie, wat postiewe resultate gelewer het. Die teenliggame teen anti-p62 besit dieselfde spesifisiteit as die kommersiële teenliggame en anti-p62 teenliggame kan dus gebruik word as waardevolle gereedskap vir toekomstige studies van mitofagie in MEF selle. Weens die feit dat hierdie teenliggame, wat gebasseer is op die ooreenkoms van epitoop volgordes, spesifiek was vir p62 van Nothobranchius epitope, is hierdie teenliggame van besonderse waarde vir immunofluoressensie studies van mitofagie in Nothobranchius in die toekoms. Die ondersoek na mtDNA-agteruitgang in Nothobranchius-vis is bewerkstellig deur PKR-analise met verskillende inleier pare van verouderende visspesies Nothobranchius korthausae en Nothobranchius guentheri, in vergelyking met toepaslike kontrolemonsters. Mitochondriale agteruitgang gebasseerd op afname in die amplifisering van groot fragmente van mtDNA van verouderende visse was nienie waargeneem nie en vereis ´n verdere en meer diepgaande ondersoek.af_ZA
dc.description.versionMastersen_ZA
dc.format.extent100 pages : illustrations (some color)en_ZA
dc.identifier.urihttp://hdl.handle.net/10019.1/127307
dc.language.isoEnglishen_ZA
dc.publisherStellenbosch : Stellenbosch Universityen_ZA
dc.rights.holderStellenbosch Universityen_ZA
dc.subject.lcshMitophagyen_ZA
dc.subject.lcshParkinson's disease -- Animal modelsen_ZA
dc.subject.lcshNeurodegenerative diseasesen_ZA
dc.subject.lcshMitochondrial DNAen_ZA
dc.subject.nameUCTDen_ZA
dc.titleStudies of mitophagy in mouse and fish models using antibodies and PCRen_ZA
dc.typeThesisen_ZA
Files
Original bundle
Now showing 1 - 1 of 1
Thumbnail Image
Name:
leukes_studies_2023.pdf
Size:
2.4 MB
Format:
Adobe Portable Document Format
Description:
Download
Collections
Masters Degrees (Biochemistry)