Theses (MSc in Molecular Medicine)

Emerging evidence of meta-analyses suggested that periodontal disease (PD) mediated by oral bacteria may increase the risk of developing neurodegenerative disorders. However, the findings were inconsistent and inconclusive. An umbrella review makes it feasible to summarise the current evidence from the published meta-analyses on this topic and enable the ranking of evidence. In addition, among periodontal pathogens, Porphyromonas gingivalis produces a calcium-independent peptidylarginine deiminase enzyme (PPAD) that induces hypercitrullination, a process that has been proposed to contribute to the onset of neurodegenerative disorders. Nonetheless, PPAD inhibitors have been scarcely reported in the literature, highlighting a significant gap in the current body of research. Therefore, this study aimed to (i) conduct an umbrella review to summarise the associations between oral bacterial diseases and neurodegenerative disorders and assess the credibility of evidence supporting such associations and (ii) evaluate the binding interactions and identify potential inhibitors of PPAD through molecular docking simulations, using the 15 compounds previously identified via 2D ligand-based similarity screening. An umbrella review of 6 eligible meta-analyses including 17 associations was conducted using predefined eligibility criteria and the credibility of evidence was graded according to AMSTAR-2 methodological quality assessment. In parallel, molecular docking simulations were performed to examine binding affinity and key residue interactions of 15 compounds within the PPAD binding site. Nine of 17 associations (52.9 %) showed statistical significance at P ≤ 0.05 based on random-effects models between PD and neurodegenerative disorders. Twelve out of the 17 associations (70.6%) had considerable heterogeneity (I2 > 50%), suggesting that the pooled estimates should be interpreted carefully. Only one association from 17 associations showed a small-study effect. Docking analyses revealed that several compounds exhibited favourable binding affinity within the PPAD binding site interacting with essential residues, particularly (4S)-5-[[(2S)-1-[[(2S)-1-Amino-3-[4-[difluoro(phosphono)methyl]phenyl]-1-oxopropan-2-yl]amino]-3-[4-[difluoro(phosphono)methyl]phenyl]-1-oxopropan-2-yl]amino]-4-benzamido-5-oxopentanoic acid exhibiting binding score of -9.737 kcal/mol. Notably, N-acetyl-L-arginine was estimated to interact directly with His236 of the catalytic triad, suggesting potential inhibition. Collectively, the umbrella review and molecular docking simulations support a biological plausible, though not definitive, link between PD and neurodegenerative disorders. Further longitudinal studies and experimental enzymatic and cellular evaluations are necessary to enhance PPAD-target inhibitor development toward clinical application.

Colorectal Cancer (CRC) is among the foremost causes of cancer-related mortality in Malaysia and worldwide. This warrants the continuous development of novel, efficient and safe therapeutic options. Glucomoringin (GMG) is a secondary metabolite derived from the plant, Moringa oleifera Lam, which is highly known for its nutritional, medicinal and environmental benefits. Preclinical studies have identified promising anticancer properties from extracts of M. oleifera and the associated secondary metabolites. Hence, this study aims to investigate the anticancer potential activity of GMG against human colorectal cancer cell lines (HCT116, HT29, HCC2998 and SW48). The cytotoxic effects of GMG were evaluated through cell viability using the MTT assay at incubation time periods of 24, 48 and 72 hours, with doxorubicin as the standard chemotherapeutic drug. Changes in nuclear morphology and apoptotic features were also assessed through Hoechst 33342 staining and fluorescence microscopy following treatment with GMG. It was observed that GMG manifests a dose-and time-dependent cytotoxicity across all colorectal cancer cell lines, with HCC2998 and SW48 showing the most significant reduction in cell viability, post treatment. GMG-treated cells displayed intensified nuclear fluorescence and irregularity in nuclear morphology, which is consistent with apoptotic cell death features. However, despite reports indicating GMG as a potent anticancer agent, our findings shows that GMG has limited antiproliferative effects, even with an IC50 value of more than 100 μM and prolonged exposure of 72 hours. Additionally, its anticancer activity is also markedly lower compared to that of the standard chemotherapy drug, suggesting limited therapeutic potential in its natural form. Nonetheless, these observations provide significant insights into the limitations of GMG and highlight important future research directions aimed at enhancing and unlocking its therapeutic potential.

Pancreatic cancer (PC) remains a highly lethal malignancy with limited therapeutic options, highlighting the need for alternatives with selective anticancer activity. Syzygium aqueum, a phytochemical-rich tropical plant, exhibits diverse bioactivities, yet its anticancer effects against PC have not been investigated. This study assessed the cytotoxic, antiproliferative, and apoptogenic effects of ten S. aqueum leaf and stem extracts, including hexane, chloroform, ethyl acetate, methanol, and aqueous extracts, on BxPC-3 cells, with selectivity evaluated in non-cancerous 3T3 fibroblasts. Cytotoxicity was assessed by MTT assays at 6.25–200 μg/ml over 24, 48, and 72 hours. Antiproliferative and apoptotic effects were assessed by doubling time analysis and Hoechst 33342 staining, respectively, with all experiments performed in triplicate. At 72 hours, leaf ethyl acetate (LE), stem aqueous (SA), leaf chloroform (LC), and stem ethyl acetate (SE) showed pronounced cytotoxicity, with IC50 values of 49.3, 50.08, 61.23, and 71.80 μg/ml, respectively, and the 72-hour time point was selected for downstream analyses due to consistent IC50 values. Stem hexane (SH) displayed the highest selectivity index exceeding 3, indicating predominantly cancer-specific cytostatic effects. Doubling time analysis demonstrated significant (p < 0.05), dose-dependent proliferation delays for all extracts except leaf aqueous (LA) and LE, whereas Hoechst staining revealed significant increases (p < 0.05) in apoptotic cells across all treatments except SH, which showed minimal apoptosis consistent with its cytostatic profile. Collectively, these findings demonstrate that S. aqueum extracts exhibit promising anticancer activity against PC cells through combined cytotoxic, cytostatic, and pro-apoptotic effects, supporting further mechanistic investigation and therapeutic development.

Parkinson’s disease (PD) is a chronic neurodegenerative disorder characterised by the progressive loss of dopaminergic neurons in the substantia nigra. Current therapies for PD focus only on symptomatic relief and do not halt disease progression. Therefore, neural stem cell transplant has emerged as a promising cell therapy option for PD, as it has the potential to restore dopaminergic neurons. However, the survival of transplanted neural stem cells within the diseased brain remains a major challenge. Salsolinol (SAL), an endogenous and exogenous neurotoxin, has been associated with PD due to its ability to generate oxidative stress and disrupt mitochondrial function in dopaminergic neurons. However, limited research has explored its impact on neural stem cells (NSCs) and the role of autophagy in mediating this toxicity. This study investigates whether SAL exerts toxic effects on NE-4C neural stem cells through autophagy-related mechanisms. NE-4C cells were exposed to different concentrations of SAL for 24, 48, and 72 hours. Cell viability was assessed using the MTT assay to determine the IC25, IC50 and IC75. Acridine orange staining was used to detect acidic vesicle formation, indicative of autophagic activity. Expression levels of autophagy-related proteins (Atg12, Beclin-1, and LC3 A/B) were measured using cell-based ELISA. Exposure to SAL resulted in a significant, time- and dose-dependent reduction in cell viability. A significant increase in acidic vesicle formation was observed after SAL treatment, suggesting enhanced autophagic activity. Additionally, ELISA results showed elevated expression of Atg12 and Beclin-1, and significant upregulation of LC3 A/B, supporting the activation of autophagy in response to SAL treatment. These findings confirm that SAL induces autophagy-mediated neurotoxicity in NE-4C NSCs, providing insights into how endogenous neurotoxins may compromise stem cell viability in PD and suggest that regulating autophagy may help improve survival of endogenous and transplanted NSCs in PD patients.

Objectives: To fabricate carbon quantum dots (CQDs) and evaluate its effects on structure and crystal orientation and mechanical, and antibacterial properties of enamel. Materials and Methods: Enamel specimens of 4 mm x 4 mm x 3 mm were cut from extracted teeth. study. CQDs were prepared using ammonium citrate and sodium hydroxide solution dialysed in deionised water. After microwave reaction, Teflon tubes were left in machine for 30 minutes to cool. Enamel block specimens were divided into 4 groups (salineS, *CQD0.1%-, **CQD0.2%-, ***CQD0.3%-, and *****CQD0.5%-) in each of the treatment solutions, soaking for about 2 weeks. X-ray diffraction analysis (XRD) was performed on enamel to determine density functional theory (DFT) calculations. Transmission electron microscopy (TEM) and zeta potentials (ζ) were performed for CQDs. After modification, enamel was evaluated using Raman mapping, TEM, and Xray diffraction (XRD) analysis. Mechanical and antibacterial properties were evaluated using atomic force microscopy and TEM/SEM. For statistical analysis, null hypothesis was set at p<0.05 probability level. Results: Discrepancies in peak position between experimental and theoretical XRD diffraction patterns are due to differences in calculated parameters of ***CQD0.3%- and ***CQD0.5%- groups bringing conformational changes. Zeta potential values were least for salineS and maximum for *****CQD0.5%-. **CQD0.2%- group exhibited reduced v₁PO₄³⁻ intensity, with *****CQD0.5%- demonstrating the highest. The groups, arranged by ascending intensity, are salineS <*CQD0.1%-, <**CQD0.2%-, <***CQD0.3%-, < *****CQD0.5%-. TEM indicated spherical carbon dots with increase in enamel lattice parameter relative to the HAP unit cell in *****CQD0.5%- groups. *****CQD0.5%- group had highest elastic modulus and nano hardness. Bond length and angles show higher significant measures in CQD groups. CQD groups demonstrated higher antibacterial activities engulfing bacteria.