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    The roles of miRNA-128 and miRNA-223 in cholesterol-mediated drug resistance in breast cancer
    (2024) Palma, Gabriella Bianca Henriques
    Breast cancer (BC) is the most prevalent cancer in women, with 70% of BC cases being hormone responsive (estrogen receptor positive (ER+)). This ER+ BC subtype relies on estrogen for enhanced cell proliferation and survival. The main therapeutic strategy to prevent hormone responsive BC recurrence is with the use of endocrine therapy such as Tamoxifen (TAM). Despite the success in reducing mortality rates of BC patients with the use of adjuvant TAM and chemotherapy, cancer drug resistance remains a significant challenge. A major contributor to this resistance is the dysregulation of cholesterol homeostasis in these cells. BC cells have elevated intracellular cholesterol levels, which is associated with cancer progression. In our previous research, we observed that the use of a cholesterol depleting agent, Acetyl Plumbagin (AP) in combination with TAM, led to the induction of cell death via cholesterol depletion. These results therefore warranted further investigation into the molecular mechanisms in which TAM + AP are involved in. MicroRNAs (miRNAs) regulate cholesterolrelated and cancer drug resistance pathways, and the aberrant expression of these miRNAs are often associated with increased cancer proliferation and resistance. It was therefore predicted that manipulating the expression of these target miRNAs could lead to a reduction in BC related drug resistance via cholesterol depletion. Thus, we aimed at investigating the roles of miRNA128 and miRNA-223 in cholesterol-mediated TAM resistance. Three BC cell lines (MCF-7 (estrogen-dependent), MDA-MB-231 (estrogen-independent), and Long-Term Estrogen Deprived (LTED)) were treated with a combination of 1 µM TAM and 10 µM AP following transfection with a miR-128 inhibitor or a miR-223 mimic. Cell viability and cholesterol levels were assessed following treatments. In addition, gene and protein expression levels involved in cancer drug resistance and cholesterol homeostasis were also assessed. It was found that the combination treatment with altered miRNA expression led to reduced cell viability and proliferation due to a reduction in free cholesterol, cholesteryl esters, and lipid rafts in all three BC cell lines. Moreover, miR-128 inhibition lowered the expression of genes involved in cholesterol synthesis and transport (HMGCR, HMGCS1, SREBF1/2, CETP, LCAT, and LDLR), drug resistance (ABCC5 and UGCG), and cell signalling (ESR1, EGFR, and IGF1R) in MCF7 cells. Whereas overexpression of miR-223 led to decreased expression in EGFR, ESR1, ABCC5, CETP, LCAT, LDLR, HMGCR, SREBF1, and SREBF2, with increased expression in ABCG1, PTEN, and TP53 in MDA-MB-231 cells. Therefore, the current study demonstrated that miR-128 and miR-223 could be potential targets in reducing TAM resistance through the depletion of excess cholesterol.
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    Interactions of chloroquine and/or ethanol on renal morphology of normal or low protein fed male sprague dawley rats
    (2018) Abdulkadir, Abdurrahman
    The aim of this study was to investigate the renal morphometric and histopathological changes, arising from the hazard of concurrent administration of chloroquine and alcohol on a background protein malnourished state using rat as experimental animal model. Sixty-four (64) rats were randomly distributed into eight groups of eight rats each as follows: control groups that received 0.9% saline and plain drinking water while on either normal or low protein diets (NPC, LPC). Chloroquine treatment groups while on either normal or low protein diets (NPQ, LPQ). Alcohol treatment groups while on either normal or low protein diets (NPE, LPE). Chloroquine and Alcohol treatment groups on normal or low protein diets (NPQE, LPQE). Chloroquine in 0.9% normal saline was administered once weekly to NPQ, LPQ, NPQE, and LPQE. While NPE, LPE, NPQE and LPQE received 6% ethanol in drinking water ad libitum. NPC and LPC received 0.9% normal saline and plain drinking water. After 8 weeks (60 days), the rats were terminated, kidneys were harvested and fixed for analyses. Routine H&E histology, Masson’s trichrome for collagen, kidney volume estimation, relative medullary thickness measurements, renal cortical thickness measurements, microscopic morphometry, glomeruli count and immunofluorescence for aquaporin 2 (AQP2) were conducted. Urine volume estimation, serum urea and creatinine were also assessed. The results showed a decreased mean body weight (p=0.0001), relative kidney weight (p=0.0001) and kidney volume (p=0.0001) in all low protein treated rats group compared to NPC and compared to their cohorts in normal protein group. The urine output decline with derangements of serum urea and creatinine in the low protein treated rats compared to NPC and compared to their cohorts in the normal protein group. There was upregulation of AQP2 water channel with increased collagen fibre deposition and distortion of normal renal histology in the experimental rat groups both within the normal protein and the low protein groups compared to NPC. In conclusion, concurrent administration of chloroquine and alcohol causes distortion of kidney histology and derangements of renal function in low protein fed rats and has the potential to cause kidney failure.
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    The total synthesis of 5-methoxy-3,4-dehydroxanthomegnin, dehydroherbarin lactone and 6-ferrocenyl-3,4-dihydroisochromene
    (2018) Sumani, Jimmy Yaphet Ephet
    Pyranonaphthoquinones, especially those with lactone functionality, are a class of naturally occurring compounds that exhibit a wide range of biological properties. For example, studies have shown that 10-hydroxy-5,7-dimethoxy-3-methyl-1H-naphtho-[2,3-c]-pyran-1,6,9-trione has a plethora of biological activities which includes, among others, cytotoxicity against McCoy cell lines and significant antibacterial property against Helicobacter pylori. This PhD project describes the total synthesis of 7,9-dimethoxy-3-methyl-1Hbenzo[g]isochromene-1,5,10-trione from simple starting materials in 14 steps in an overall percentage yield of 5%. The synthesis was achieved by subjecting the commercially available, 2,4-dimethoxybenzaldehyde, to a number of synthetic transformation protocols we have developed in our laboratory group. The key steps in the synthesis included the PIFA mediated methoxylation of the naphthalene nucleus that was constructed from the Stobbe condensation reaction and the palladium assisted Wacker oxidation reaction of the naphthoic acid intermediates. We also successfully synthesized 10-hydroxy-5,7-dimethoxy-3-methyl-1Hbenzo[g]isochromene-1,6,9-trione starting from 2,4,5-trimethoxybenzaldehyde in 15 steps in an overall percentage yield of 1%. This was achieved by employing the protocol we established during the synthesis of 7,9-dimethoxy-3-methyl-1H-benzo[g]isochromene-1,5,10trione. In the course of achieving the total syntheses of 7,9-dimethoxy-3-methyl-1Hbenzo[g]isochromene-1,5,10-trione and 10-hydroxy-5,7-dimethoxy-3-methyl-1Hbenzo[g]isochromene-1,6,9-trione a number of unexpected products were isolated and characterized. The second part of the project was aimed at developing a methodology for coupling a ferrocene moiety to 3,4-dihydroisochromene and subjecting the resultant ferrocenyl 3,4dihydroisochromene to oxidative demethylation protocols. Ferrocene coupled hybrid compounds are currently receiving a lot of attention because of their application in medicine. Abstract iii For example, ferroquine is active against both chloroquine-sensitive and chloroquine-resistant Plasmodium falciparum strains. The synthesis of 6-ferrocenyl-(±)-trans-3,4-dihydro-5,8-dimethoxy-1,3-dimethyl-6-vinyl-1Hisochromene was achieved starting from 2,5-dihydroxyacetophenone in 10 steps in an overall percentage yield of 10%. The key steps in this methodology involved the base mediated ring closing reaction which delivered (±)-trans-3,4-dihydro-5,8-dimethoxy-1,3-dimethyl-6-[(E)-1propenyl]-1H-isochromene and the McMurray cross-coupling metathesis reaction which afforded 6-ferrocenyl-(±)-trans-3,4-dihydro-5,8-dimethoxy-1,3-dimethyl-6-vinyl-1Hisochromene. The oxidative demethylation of 6-ferrocenyl-(±)-trans-3,4-dihydro-5,8-dimethoxy-1,3dimethyl-6-vinyl-1H-isochromene to complete the total synthesis of 6-ferrocenyl-(±)-trans3,4-dihydro-1,3-dimethyl-6-vinyl-1H-isochromene-5,8-dione was unsuccessful and an alternative protocol was developed which was aimed at coupling of ferrocene moiety to (±)trans-6-bromo-3,4-dihydro-1,3-dimethyl-1H-isochromene-5,8-dione at the last step. This methodology did not yield the target molecule, 6-ferrocenyl-(±)-trans-3,4-dihydro-1,3dimethyl-6-vinyl-1H-isochromene-5,8-dione. However, a number of potentially biologically important serendipitously obtained compounds were isolated. The last part of this PhD project involved in vitro biological screening of 3 selected compounds synthesized in this project against the Gambian FCR-3 strain of Plasmodium falciparum. All the 3 compounds namely, (±)-trans-15-bromo-5,13-dimethoxy-2,4,10,12tetramethyl-4,7,9,10,12,13-hexahydro-1H-7,13-methanopyrano[4',3':6,7]oxocino[2,3-f]isochromen-8(2H)-one, 3-(chloromethyl)-3,4-dihydro-5,7,10-trimethoxybenzo[g]isochromene1,6,9-trione and bis[(±)-trans-3,4-dihydro-8-methoxy-1,3-dimethyl-1H-isochromen-5yloxy]methane showed promising activity. (±)-Trans-15-bromo-5,13-dimethoxy-2,4,10,12tetramethyl-4,7,9,10,12,13-hexahydro-1H-7,13-methanopyrano[4',3':6,7]oxocino[2,3-f]isochromen-8(2H)-one showed the best activity with an IC50 value of 0.0018 µM. This value was better than the activity shown by the reference compounds, quinine and dihydroartemisinin, which showed activity of 2.81 µM and 0.0061 µM, respectively, under the same assay conditions.
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    The pharmacokinetics and pharmacodynamics of kanamycin and capreomycin in patients with drug resistant-tuberculosis and the relationship between hearing levels: a feasibility study
    (2018) Hollander, Cara
    Individuals respond differently to medication as a result of their genetic inheritance. These differences can result in the under- or over-dosing of medication, which may affect the efficacy or in the case of aminoglycosides (kanamycin) and polypeptides (capreomycin), result in toxicity. In South Africa, administration of the standardised Drug Resistant -Tuberculosis (DR-TB) medication regimen is simplified across four weight bands. These bands accommodate the formulations available in the country while complying with international requirements for minimum, maximum and average dose per kilogram. There is a dearth of information on the ideal concentration, pharmacokinetics, and pharmacodynamics of kanamycin (KM) and capreomycin (CM) in patients with DR-TB and relationship of this on hearing levels. Thus, this study aimed to establish the feasibility of investigating the pharmacokinetics and pharmacodynamics of kanamycin and capreomycin in patients with DR-TB and the relationship between hearing levels. This feasibility study employed a prospective, cross-sectional, exploratory, descriptive and case series research design. A total of 22 participants (mean age 33.78 years, ±7.3) participated in this multi-site study at Helen Joseph (HJH) and South Rand Hospitals (SRH). The majority of the paryicpants were females (68%, n=15). Participants underwent audiological (otoscopy, tympanometry, ultra high frequency DPOAEs, ultra high frequency pure tone audiometry) and pharmacological assessments at baseline and every two weeks for the first three months of treatment. Creatinine clearance was measured, and the overall outcome of treatment was evaluated in relation to the pharmacokinetics. Results revealed high-frequency hearing loss with both kanamycin and capreomycin, specifically in the ultra-high frequencies (9kHz to 16kHz). Clinically significant ultra-high frequency loss noted was with pure tone audiometry from week four after the initiation of treatment, and from week six in the high frequencies (6kHz to 8kHz). Pharmacokinetic measurements showed erratic levels of kanamycin and capreomycin, with considerable differences among individuals, specifically with the peak readings. Mean peak levels for kanamycin were within the target range yet were subtherapeutic for the capreomycin participants. Kanamycin also correlated to more reduced kidney function when compared to capreomycin. Participants’ culture converted within the first two months from baseline, however, long-term culture results are unknown. Trough levels were also below 10 μg/ml and not within a toxic range, despite the hearing loss detected. This research identified many challenges with regard to establishing the feasibility of investigating the pharmacokinetics and pharmacodynamics of kanamycin and capreomycin in patients with DR-TB and the relationship between hearing levels. Participant enrolment was poor, with high attrition. This study also highlighted the need for a standardised ototoxicity monitoring protocol designed for this population which led to the development of ‘Ototcalc’: an ototoxicity calculator in the form of a mobile application designed to assist healthcare professionals in the classification of significant ototoxicity as well as with management recommendations. With the considerations identified in this study to further enhance the feasibility, the pharmacokinetics and pharmacodynamics of kanamycin and capreomycin are recommended for further exploration in relation to toxicity and efficacy with a larger sample, combined with the use of ‘Otocalc’.