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Doctoral Degrees ( Medical Science)

Permanent URI for this collectionhttps://hdl.handle.net/10413/14011

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    An investigation into kojic acid-associated mitochondrial toxicity and inflammation in melanoma cells (SK-MEL-1) = Uphenyo ku-esidi yekojiki ehlobaniswa nokukhinyabezeka kwemayithokhondriya nokuvuvukala ezinhlayiyeni zemelanoma (SK-MEL-1).
    (2023) Suritham, Tamzin Kimera.; Chuturgoon, Anil Amichund.; Ghazi, Terisha.
    ojic acid (KA), 5-hydroxy-2-(hydroxymethyl)-4H-pyran-4-one, is used in agriculture, food, and cosmetics. KA is known to have antimicrobial, antifungal, antioxidant, and anti-inflammatory properties. The cosmetic industry's increasing interest in KA is due to its ability to inhibit tyrosinase activity resulting in skin lightening. The mitochondria play a key role in maintaining homeostasis and ensuring efficient melanin production. Therefore, mitochondrial dysfunction has severe effects on the skin. This study investigates mitochondrial stress, antioxidant responses, protein kinase signalling and inflammation in human melanoma (SK-MEL-1) cells. The mitochondria are important in processing metabolites and supplying the cell with energy in the form of ATP. KA interacts with key mitochondrial homeostasis proteins. Our results found an increase in macromolecule damage specifically lipid peroxidation and protein oxidation. Due to oxidative conditions, increased Nrf2 expression was observed. LON protease is ATP-dependent and regulated by Sirtuin 3 expression. Mitochondrial function was affected illustrated by decreased ATP production leading to decreased LON protease and Sirtuin 3 protein expression. Following increased oxidative stress, KA suppressed the expression of protein kinases but increased inflammatory mediators. There was decreased expression of phospho-Akt, Akt, phospho-GSK3β, p38 and ERK1/2. The mediation of the NLRP3 inflammasome involves priming and activation. At concentrations with high proliferation, NFκB gene and protein expression was activated. The protein kinase signalling pathways are known as mediators of inflammation; however, protein and gene expression of inflammatory mediators was increased following KA treatment. The inflammasome was subsequently activated as shown by an increase in intracellular caspase 1 levels as well as NLRP3, ILβ and IL-6 expression. KA induced mitochondrial stress and suppressed mitochondrial homeostasis proteins. The increased Nrf2 expression could have further downregulated LON protease expression and increased macromolecule damage. Oxidative conditions could have activated the inflammasome pathway independent of protein kinase signalling. In conclusion, KA displayed mitochondrial toxicity following acute exposure by suppressing mitochondrial homeostasis, protein kinase pathways and initiating inflammation. Iqoqa. I-esidi yeKojic (KA), 5-hydroxy-2-(hydroxymethyl)-4H-pyran-4- eyodwa, iyasetshenziswa kwezolimo, ekudleni nasezimonyweni. I-KA yaziwa ngokuba ne-antimicrobial, antifungal, antioxidant, nezinto ezibanga ukuvuvukala. Luyakhula uthando lwezimboni zezimonyo ekuthandeni i-KA ngenxa yobukhona bayo ukuphazamisa ukusebenza kwetyrosinase okuholela ekutheni isikhumba sibe mhlophe. Kunokumqoka kakhulu okwenziwa imayithokhondriya ekugcineni usimamisoluzinzo lobunjalomzimba nokuqinisekisa ukukhiqizwa okwanele kwemvikelambala, imelanin. Ngakho ke, ukungasebenzi kwemayithokhondriya kunemithelela emibi esikhunjeni. Lolu cwaningo luphenya ngengcindezi yemayithokhondriya, ukusebenza kwe-antioxidant, ukukhombisa iphrotheyini khinasi nokuvuvukala kwezinhlayiya (SK-MEL-1) zemelanoma yomuntu. Imayithokhondriya ibalulekile ekuqhubeni umsebenzi wokugaya ukudla nokunika inhlayiya amandla ayisimo se-ATP. I-KA iyahlangana namaphrotheyini asemqoka osimamisoluzinzo lobunjalomzimba bemayithokhondriya. Imiphumela yethu yathola kukhula ukulimala emolekhiyulini enkulu okuyiliphidi iphreroksideyishini nokuncipha kwezinhlayiyabugesi ezihambayo zamaphrotheyini. Ngenxa yezimo zokuncipha kwezinhlayiyabugesi ezihambayo, kwabhekwa ukukhula kokuziveza kweNrf2. Okubalulekile empilweni nasekusebenzeni kwezinhlayiya, kuyi-LON, I-LON phrothizi kuncike kuyi-ATP futhi kulawulwa ukuziveza kweSirtuin 3. Umsebenzi wemayithokhondriya kwaphazamiseka kwaboniswa ukuncipha komkhiqizo we-ATP okwaholela ekuncipheni kweLON phrothizi nokuziveza kwephrotheyini iSirtuin 3. Ukulandela ukukhula kwengcindezi yokuncipha kwezinhlayiyabugesi zamaphrotheyini, i-KA yacindezela ukuziveza kwephrotheyini yekinases kodwa kwakhulisa ukuvuvukala kwezixhumanisi zengxube ezihlanganisayo. Kwaba nokuncipha kokuziveza kwephospho-Akt, i-Akt, i-phospho-GSK3β, i-p38 a ne-ERK1/2. Isihlanganisi seNLRP3 inflammasome sibandakanya ukulungiselela nokukhuthaza. Kusilinganisobungako esineproliferation ephezulu, ukuziveza kofuzo lweNFκB kanye nephrotheyini kwakhuthazeka. Ukukhombisa izindlela kwephrotheyini ikinase kwaziwa ngokuthi ukuvuvukala kwezixhumanisi; nokho, ukuziveza kwezixhumanisi zamaphrotheyini nawofuzo avuvukele kwakhula kulandela ukwelashwa kweKA. I-inflammasome yakhuthazwa njengoba yayiboniswa ngokukhula kwamazinga loku-1 lezinhlayiya zokufanayo kwecaspase nokuziveza kweNLRP3, i--β ne- IL-6. I-KA iletha ingcindezi yemayithokhondriya ibuye icindezele amaphrotheyini osimamisoluzinzo lobunjalomzimba bemayithokhondriya. Ukukhula kokuziveza kweNrf2 bekungeza ukwehlisa ukulawulwa kokuziveza kweLON phrothizi nokukhula kokulimala kwemolekhiyuli enkulu. Izimo zokuncipha kwezinhlayiyabugesi ezihambayo ngabe kukhuthaze izindlela ze-inflammasome ezizimele ezikhombisa iphrotheyini yekhinasi. Ucwaningo lwaphetha ngokuthi i-KA yabonisa ubuthi bemayithokhondriya kulandela ukungavimbeki kwabo okunamandla ngokuthi bucindezele usimamisoluzinzo lobunjalomzimba lwemayithokhondriya, izindlela zephrotheyini khinasi nokuqala kokuvuvukala.
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    Drug transporter expression and genetic polymorphisms in HIV endemic settings = Indlela yokuthutheka kwemithi emzimbeni nesakhiwo esingxube sofuzo ezimweni zokubhebhetheka kwesandulela ngculazi.
    (2023) Zondo, Nomusa Margaret.; Archary, Derseree.; Sobia, Parveen.
    Pre-exposure prophylaxis (PrEP) in the form of oral Truvada® remains the standard of care for HIV prevention in South Africa. Despite the availability of PrEP, HIV infections continue in young women significantly more than in men. Clinical trials testing antiretrovirals containing tenofovir as topical or oral PrEP formulations in African women, produced inconsistent patterns of efficacies against HIV. Effectiveness of oral and topical PrEP is dependent on adequate drug delivery and availability to cells and tissues targeted by HIV. Our study, therefore, focused on how different biological factors: drug transporter expression, single nucleotide polymorphisms (SNPs) in drug transporter genes and genital inflammation modulate PrEP disposition in African women. We characterized drug transporter mRNA expression in two compartments, the female genital tract (FGT) and blood, at baseline, 3 and 6 months in 45 women taking oral PrEP-Truvada®. Additionally, the impact of SNPs in 393 women and genital inflammation in 45 women on circulating tenofovir and drug transporter mRNA expression were determined. SNPs in drug transporter genes: ABCB1 3435G>A; ABCC1 198217T>C; ABCC2 1249G>A; ABCC4 3463T>C; ABCC4 4131A>C and ABCC4 4976A>G were evaluated using real-time PCR. mRNA expression of efflux P-gp, MRP-2, MRP-4, MATE-1 and influx OAT-1 and OAT-3 drug transporters was evaluated using quantitative real-time PCR. Genital inflammation was measured in cervicovaginal specimens using a 28-cytokine multiplexed platform. Results showed that ABCC4 4976A>G and ABCC4 3463T>C SNPs alter circulating tenofovir differently. While the ABCC4 4976A>G SNP significantly increased the mRNA expression of the ABCC4 gene (p=0.0132), there was inverse association with circulating tenofovir (p=0.018). In contrast, although the ABCC4 3463T>C SNP did not significantly impact mRNA expression of the ABCC4 gene, it was significantly and directly associated with circulating tenofovir (p<0.05). Correlation analyses showed moderately significant associations between the mRNA expression of the influx drug transporter OAT-1 in the FGT and blood pre- and post- PrEP exposure (rs<1, p<0.05). In contrast efflux drug transporters P-gp, MATE-1, MRP-2 and MRP-4 showed significance after PrEP initiation (3 and 6 months) (rs<1, p<0.05). For pro-inflammatory cytokines, linear mixed models showed negatively correlated trends between IL-1β and MCP-1 and influx drug transporter OAT-1 and OAT-3 (p<0.1), while IL1Rα and TNF-α showed these correlations with efflux drug transporters MRP-2 and MRP-4 (p<0.1). Collectively our results suggested that PrEP disposition can be modified through a convergence of host genetics and different biological factors: drug transporter expression, SNPs in drug transporter genes and inflammation. Findings from such studies may be used to better understand PrEP pharmacokinetics and aid in the implementation of optimal PrEP dosages. This 14 will ultimately inform on effective and safe PrEP for HIV prevention especially in vulnerable and at-risk African women. Iqoqa. ENingizimu Afrika ipre-exposure prophylaxis (PrEP) oral-Truvada® kuseyiyona ndlela yokunakekela ekuvimbeleni isandulela ngculazi, i-HIV. Kodwa ukutheleleka nge-HIV kuyaqhubeka kakhulu kwabesifazane abasebancane. Ukuvivinywa kwemithi lapho kuhlolwa i-PrEP egcotshwayo/neyasemlonyeni kulabo besifazane abangama-Afrika kuveza izimo ezingazinzile ekulweni ne-HIV. Ukuphumelela kwale mithi egcotshwayo/neyasemlonyeni kuncike kakhulu ekuhambisekeni komuthi okwanele nasebukhoneni bama-cells aqondwe ngqo yi-HIV; izinto ezihambisana nomzimba ezibalulekile zibonakele ekulawuleni ukusebenza kwe-PrEP ezindaweni ezithelelekile. Lolu cwaningo, lwabheka ukuthi izimo zomzimba: indlela yokuthuthwa komuthi emzimbeni, isakhiwo esisodwa ekuthuthweni komuthi emzimbeni isingle nucleotide polymorphisms (SNPs) kanye nokuvuvukala kwezitho zangasese kunomthelela muni ekusebenzeni kwePrEP kwabesifazane abangama-Afrika. Kwakhethwa isithuthi semithi i-mRNA-expression ngezindlela ezimbili: umgudu wezitho zangasese kwabesifazane, ifemale genital tract (FGT) kanye negazi, ekuqaleni kocwaningo, izinyanga ezi-3 neziyi- 6 kwabesifazane abangama-N=45 abathatha i-PrEP-Truvada® ngomlomo. Kwabe sekubhekwa i-mRNA-expression ye-P-gp, MRP-2, MRP-4, MATE-1 kanye ne-OAT-1 kanye ne-OAT-3 okuyizithuthi zemithi ezahlolwa kusetshenziswa ucwaningozibalo lwereal-time-PCR. Umthelela we-SNPs (N=393) kanye nokuvuvukala kwezitho zangasese (N=45) ekuzungeziseni i-tenofovir kanye nesithuthimithi i-mRNA-expression kwabonakala. Ukubonakala kwe-SNPs kwizithuthimithi zofuzo: ABCB1[3435G>A]; ABCC1[198217T>C]; ABCC2[1249G>A]; ABCC4[3463T>C]; ABCC4[4131A>C] kanye ne-ABCC4[4976A>G] kwahlolwa kusetshenziswa ireal-time-PCR. Ukuvuvukala kwezitho zangasese kwakalwa kumasampula ayethathwe ebuntwini babesifazane kusetshenziswa indlela ye-28-cytokine-multiplexed platform. Imiphumela yakhombisa ukuthi i-ABCC4[4976A>G] kanye ne-ABCC4[3463T>C] SNP ishintsha ukuzungeza kwetenofovir ngendlela ehlukile. I-ABCC4[4976A>G] SNP yakhulisa kakhulu i-mRNA-expression ye-ABCC4 gene (p=0.0132), kodwa yanomthelela ophambene ekuzungezeni kwetenofovir (p=0.018). Uma kuqhathaniswa, yize i-ABCC4[3463T>C] SNP ingabanga namthelela otheni kwi-mRNA-expression yofuzo i-ABCC4, yayamana kakhulu nokukhula ekuzungeziseni itenofovir (p<0.05). Ukuhlaziya kwalokhu kuhlobana kwakhombisa ukuhambisana okubalulekile phakathi kwe-mRNA-expression ye-OAT-1 kwiFGT kanye negazi ngaphambi nangemuva kokusetshenziswa kwe-PrEP. Kanti i-P-gp, MATE-1, MRP-2 kanye ne-MRP-4 kwakhombisa ukubaluleka emuva kokuqalwa kwe-PrEP ezinyangeni ezi-3 kuya kweziyi-6 (rs<1, p<0.05). Ekuvuvukaleni kwezitho zangasese, izindlela eziqondile ezingxube zakhombisa ukungahlobani phakathi kwe-IL-1β kanye ne-MCP-1 ene-OAT-1 kanye ne-OAT-3; IL-1Rα kanye ne-TNF-α ene-MRP-2 kanye ne-MRP-4 (p<0.1). Seyihlangene yonke le miphumela yakhombisa ukuthi ukusebenza kwe-PrEP kuyashintshashintsha ngokubambisana kwezimo ezahlukene emzimbeni: yindlela umuthi othutheka ngayo, ama-SNPs, kanye nokuvuvukala kwezitho zangasese. Imiphumela yalolu cwaningo ingasetshenziselwa ukuba kuqondwe kangcono amandla okusebenza kwe-PrEP kanye nokuqalwa kokukala ubungako bomuthi we-PrEP ngendlela efanele. Ngaleyo ndlela, kungaba nokusebenziseka ngempumelelo nangendlela ephephile kwe-PrEP ukuze kunqandwe isandulela ngculazi kwabesifazane bama-Afrika abasengcupheni.
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    Strategic application of in silico drug discovery approaches to discover novel TB drugs = Ukusetshenziswa komqondosu womuthi wokwelapha i-in silico ngezindlelakwenza ukuze kutholakale imithi yokwelapha i-TB emisha.
    (2023) Kisten, Kimona.; Mhlongo, Ndumiso Nhlakanipho.; Kumalo, Hezekiel Mathambo.
    Tuberculosis is one of the major causes of mortality worldwide due to the onset of bacterial infection. It remains a continuous field of study as a result of the emergence of drug resistant strains whereby first- and second-line drugs are ineffective. Variations associated with proteins in the bacteria, Mycobacterium tuberculosis, can be attributed largely to poor compliance of patients and the existence of co-morbidities within a person, inevitably leading to coinfections and a diminished immune response. This evolution of the bacteria therefore calls for extensive research to be carried out on enhanced drug design and development techniques. Re-evaluation of previously identified drug targets, determination of newly viable drug targets and the identification and design of small molecule inhibitors form a basis on the forefront of this research. Computer aided drug design techniques provide a novel method exponentially gaining popularity. Molecular modelling utilizing in silico developed methods, paired with drug repurposing, pose a viable, cost effective and efficient solution for drug design strategies. The use of chemical interaction data associated with small molecules forming complexes with drug targets of interest help understand the behaviour and mechanism of a protein before graduating to wet method techniques. Using the fundamentals of docking, molecular dynamics and virtual screening, various small molecules with the potential to provide extensive inhibition capability can be identified. This study investigates three major drug targets of Mycobacterium tuberculosis (Mtb): Enoyl-[acyl-carrier-protein] reductase (inhA), β-ketoacyl ACP synthase (KasA), and Dihydropteroate synthase (DHPS/folP1) involved in the mycolic acid and folate pathways. Various tools inclusive of gene ontology, network-based inference, virtual screening and tailored pharmacophore as a function of molecular modelling and drug repurposing were used to identify potential potent inhibitors and comprehend the understanding of the structural changes, conformations and interactions associated with the protein and the response to suggested drug hits with the potential to affect an overall protein structure in consideration with the formation of a complex. This approach can potentially serve as a platform to the development and discovery of novel drugs against a wide range of drug targets. Iqoqa. Isifo sofuba singezinye zezimbangela ezinkulu zokushona emhlabeni jikelele ngenxa yokuthathelwana kwezifo zamagciwane. Silokhu siqhubeka nokuba wumkhakha ocwaningwayo ngenxa yokuqubuka kwezinhlobo ezingezweli emithini yokwelapha lapho imithi esetshenziswe ezingeni lokuqala nelesibili ingasebenzi. Umehluko ohlobene nezakhamzimba amaphrotheyni asegciwaneni iMycobacterium tuberculosis, kungahlotshaniswa kakhulu nokuhambisana okungekuhle kweziguli nobukhona bezinye izifo kumuntu, okuholela ekuthelelekeni ngokuningi nokwenza izinga lamasosha omzimba ehle. Le nguqukomumo yegciwane ibiza ukuba kube nocwaningo olunzulu okumele lwenziwe ngohlelo lwemithi ephuculiwe namasu okuluphucula. Ukuhlola kabusha kwemithi ebihlonzwe esikhathini esedlule ukuhlonzwa kokuhloswe ngemithi yokwelapha nesakhiwo sezivimbi zamamolekhyuli amancane kuyisisekelo esiphambili kulolu cwaningo. Amasukhono emithi ehlanganiswe ngokwekhompyutha ahlinzeka indlela esazanywa enokuthandwa okukhula kakhulu. Ukuqondisa amamolekhyuli asetshenziswa ezindleleni ezakhelwe i-in silico, ahambelaniswa nokusetshenziselwa ezinye izifo kwemithi, okuqhamuka isixazululo esinezindleko eziphansi nezisebenza ngezindlela zamasu okwakha imithi. Ukusetshenziswa kwemininingo yokuxutshwa kwamakhemikhali okuhlobene namamolekhyuli amancane kwakha izinhlanganisela zemithi okusoshwe ukusiza ukuqonda insebenzo kanye nendlela yephrotheyni ngaphambi kokuba kwedlulelwe emaswini ayizindlela zokumanzisa. Kusetshenziswa izingqikithi zokuzimelelisa, amadayinamikhi amamolekhyuli nokuskrina ungekho endaweni, amamolekhyuli amancane ayizinhlobo ezehlukene ezingakwazi ukuhlinzeka ukukwazi ukuvimbela okusezingeni elikhulu okungahlonzwa. Lolu cwaningo luphenya izinhlobo ezintathu zemithi esoshelwe ukusetshenziselwa i-Mycobacterium tuberculosis (Mtb): i-Enoyl-[acyl-carrier-protein] reductase (inhA), i-β-ketoacyl ACP synthase (KasA), neDihydropteroate synthase (DHPS/folP1) efakwe emigudwini yemycolic acid nefolate. Amathuluzi ehlukene ezinsizakusebenza ezisuselwa ekucwaningeni ngobunjalobukhona bofuzo, ukuqagulwa okususelwa ebuxhakaxhakeni, ukuskrina ungekho lapho kusetshenzelwa khona nensebenzomithi ehleliwe njengokomgomo wokwakhiwa kwamamolekhyuli nokusetshenziswa ngendlela entsha kwemithi kwenziwelwa ukuhlonza izivimbinsebenzobuthi ezikhona nokuzwa ukuqonda ushintsho lokomumo, okuhambelana nokuxhumana okuhambelana namaphrotheyni nempendulo yemithi ephakanyisiwe engaba nomthelela emumweni ophelele wephrotheyni uma kubhekwa isakhiwo senkimbingxube. Leli su lingakwazi ukuba yinkundla yentuthuko nokuthola imithi esazanywa esetshenziselwa izinhloso ezehlukene zokwelapha.