Theses (MSc. Molecular Medicine)

Rheumatoid arthritis (RA) is a chronic inflammatory condition which primarily affecting the joints and is characterized by dysregulated peptidylarginine deiminase (PAD) activity. PAD enzymes play a crucial role in catalysing the citrullination process and are implicated in RA pathogenesis by modifying RA-associated autoantigens. Meanwhile, Porphyromonas gingivalis (P. gingivalis), a key bacterium responsible for periodontitis, produces P. gingivalis peptidylarginine deiminase (PPAD). PPAD serves as a critical link between periodontitis and RA by generating autoantigens that trigger immune responses in RA patients. Given its role in autoimmunity, inhibiting PPAD activity presents a promising approach for managing RA. This study aims to evaluate the in vitro inhibitory effects of five FDA-approved compounds on PPAD and to identify key structural features responsible for protease inhibition. In this study, PPAD gene was cloned into pColdI plasmid and transformed into DH5α competent cells of Escherichia coli, then PPAD were overexpressed by the induction of isopropyl-β-D-1-thiogalactopyranoside (IPTG). The overexpressed cells then were sonicated to extract the PPAD enzyme (protein). The cells then pelleted because PPAD was in soluble form, then only the supernatant subjected for the citrulline colorimetric assay. In colorimetric assay, five FDA-approved compounds which had subjected to prior structure-based virtual screening, were evaluated in this study to confirm their inhibitory activity against the PPAD. Then the compounds that have the highest ability to reduce the citrullination activity, were subjected to IC50 study in order to assess the inhibitory effects of this top compound. The result indicated that PPAD gene was successfully inserted into the pColdI plasmid and transformed into DH5α competent cells. The DH5α-pColdI-PPAD cells were successfully overexpressed and subjected to sonication for PPAD enzyme extraction. The supernatant obtained after high-speed centrifugation was used for the citrulline colorimetric assay. In this assay, five drugs (Amifostine, 4-amino-3-hydroxybutyric acid, Eflornithine, Fludarabine, and Levetiracetam) were tested at 50 μM concentration against the PPAD enzyme. Among them, only Eflornithine exhibited the highest reduction in PPAD citrullination activity, showing a 16.3% decrease. Then further IC50 analysis revealed that Eflornithine could inhibit the PPAD citrullination activity by 50%, with an IC50 value of 108.6 μM. Although Eflornithine has demonstrated potential in reducing the PPAD enzyme activity, further validation is required before Eflornithine can be considered as appropriate treatment for RA.

Excessive or dysregulated inflammation can lead to Chronic inflammatory respiratory diseases (CIRDs), such as Chronic Obstructive Pulmonary Disease (COPD) and asthma, which are significant global health concerns characterized by persistent airway inflammation and tissue damage. While conventional treatments have been effective, they are often associated with severe side effects driving the search for safer, natural alternatives. Nobiletin, a flavonoid derived from citrus peels, has emerged as a promising candidate due to its potent anti-inflammatory. It’s clinical application is hindered by poor water solubility, low oral bioavailability, and extensive first-pass metabolism. To address these limitations, this study explores the use of Nanostructured Lipid Carriers (NLCs) as an innovative mechanism of delivery for nobiletin. This research focused on formulating and characterizing nobiletin-loaded NLCs (Nob-NLC) to optimize their physicochemical properties and evaluate their efficacy in targeting respiratory inflammation using mouse macrophage cell line (RAW 264.7). The optimized formulation exhibited desired particle characteristics and a sustained release demonstrating its potential for enhanced therapeutic efficacy. In vitro studies revealed that Nob-NLC when compared to free nobiletin, demonstrated a greater anti-inflammatory action. Nob-NLC in the incubation significantly decreased the production of Nitric Oxide to 26.5 ± 0.27 μM (41.3 % decrease compared to LPS alone, p < 0.0001) and Reactive Oxygen Species 76.5% (108.5 ± 0.32 μM, p < 0.0001 vs. LPS alone). Furthermore, it reduced the mRNA expression of pro-inflammatory cytokines : reducing TNF-α expression by 69.1% (0.34 ± 0.02, p < 0.0001 vs. LPS-only group) and IL-6 expression by 42.5% (0.62, p < 0.0001 vs. LPS-treated group. The difference between action of pure nobiletin and Nob-NLC were statistically significant (p < 0.0001) in all in vitro studies except the Nitric Oxide study (p > 0.05). The results underscore the potential of nobiletin-loaded NLCs as an effective treatment strategy for managing CIRDs.

Teucrium polium is a medicinal plant widely used in traditional medicine and is known for its diverse pharmacological activities, including antioxidant, anti-inflammatory, antimicrobial, and anticancer properties. This study investigates the anticancer potential of T. polium extracts incorporated into silver nanoparticles (AgNPs). Following extraction, the total phenolic content (TPC) and total flavonoid content (TFC) of the extracts were quantified, and their chemical profiles were analyzed using UVVis spectroscopy, Fourier-transform infrared spectroscopy (FTIR), liquid chromatographymass spectrometry (LC-MS), and high-performance liquid chromatography (HPLC). Among the tested solvents, the ethanolic extract exhibited the highest TPC (140.2 ± 2.4 mg GAE/g), followed by the aqueous extract (107.2 ± 1.8 mg GAE/g), while the hexane extract contained negligible phenolic content. Consequently, only the ethanolic and aqueous extracts were utilized for AgNP synthesis. The synthesis of AgNPs was optimized by varying the extract concentration, pH, and incubation time. The most stable and optimal nanoparticles were obtained using a T. polium extract concentration of 10 mg/mL, a pH of 10.5, and an incubation period of 4 hours. These optimized AgNPs were subsequently evaluated for anticancer activity. Cytotoxicity assays demonstrated that ethanolic AgNPs exhibited higher toxicity toward Caco-2 colorectal cancer cells compared to CCD-841 normal colon cells. At the highest concentration tested (10 μg/mL), Caco-2 cell viability was significantly reduced to 11.15 ± 0.69%, whereas CCD-841 cells retained a higher viability of 58.54 ± 2.98%. At the lowest concentration (0.3125 μg/mL), ethanolic AgNPs had minimal cytotoxic effects, with Caco-2 cell viability at 59.79 ± 1.62% and CCD-841 cell viability at 92.11 ± 0.95%. These findings suggest that CCD-841 cells exhibit lower sensitivity to AgNP exposure at sub-lethal concentrations compared to Caco-2 cells, highlighting the potential selectivity of T. poliumbased AgNPs for cancer therapy.

Background: Neural tube defects (NTDs) are congenital malformations of the brain, spine, or spinal cord resulting from genetics or nutritional deficiencies in folates and other micronutrients during early embryonic development. Inadequate folate increases homocysteine, which increases the risk of NTDs. Numerous discussions and studies on the efficacy of supplementation with activated 5-Methylhydrofolate (5-MTHF) versus synthetic folic acid in improving blood folate levels in women of childbearing age. This systematic review and meta-analysis aim to evaluate the available evidence and synthesise data on the comparative impact of these two forms of folate supplementation. Methods: A systematic review and meta-analysis were conducted by PRISMA guidelines. Relevant studies were identified through electronic searches of PubMed, Scopus, and ScienceDirect, covering randomised controlled trials published up to March 2024. Inclusion criteria focus on trials comparing 5-MTHF and folic acid supplementation across various populations, including pre-pregnancy, pregnancy, postpartum, lactation, and women with MTHFR gene polymorphisms. Risk of bias was assessed using the Cochrane Risk of Bias tool, which evaluated factors such as randomisation, blinding, and completeness of outcome data. Meta-analysis was performed using Comprehensive Meta-Analysis (CMA) and RevMan software to synthesize data on plasma folate, and red blood cell folate levels. Results: The study aims to review existing evidence from a total of ten studies involving 1,351 women across various stages of pre-pregnancy, pregnancy, post-partum, and lactation, as well as women with MTHFR gene polymorphism. The methodologies included randomised, controlled trials with different dosages of folic acid, 5-MTHF, or placebos. Key biomarkers measured included plasma folate, red blood cell (RBC) folate, homocysteine, and unmetabolised folic acid levels. Results indicate variability among studies where four studies showed higher blood or plasma folate levels with 5-MTHF compared to folic acid/placebo, while three others showed similar increases in folate levels but with potential additional benefits such as reduced unmetabolised folic acid or lower homocysteine levels in the 5-MTHF groups. The remaining three studies found comparable efficacy between the two forms of folate, one of which suggested a relationship between the MTHFR (methylenetetrahydrofolate reductase) genotype and high levels of plasma homocysteine, which can contribute to various health risks. There were ten studies assessed for risk of bias. Two studies were of some concerns due to lack of clarity in the reported outcome data and the remaining eight studies have a low risk of bias. Of the eight studies included in the analysis, two studies, short-term duration of 12 weeks or less weeks, reported significant positive differences in favour of 5-MTHF while six studies, long-term duration of more than 12 weeks, favoured folic acid over 5-MTHF. Discussion: Optimal blood folate levels are important in preventing NTDs. Many variations exist in these studies that warrant further standardised research to clarify optimal dosages and durations of supplementation with 5-MTHF versus folic acid. The benefits of reduced homocysteine and unmetabolised folic acid through 5-MTHF supplementation may bring additional benefits to high-risk groups. Conclusion: This review highlights the substantial heterogeneity among studies comparing 5-MTHF and folic acid supplementation. While short-term studies favour 5-MTHF, long-term studies generally favour folic acid. These findings highlight the need for further research to standardize methodologies and clarify supplementation guidelines.

Pancreatic cancer is characterized by its aggressive metastasis and immunosuppressive microenvironment, contributing to poor therapeutic outcomes. Current strategies have had limited success, with a 5-year survival rate remaining dismal. Recent research has focused on repurposed drugs, such as geldanamycin, which has demonstrated the ability to downregulate TGFBI and C3-proteins implicated in lymphocyte suppression. However, the functional impact of this downregulation remains unclear. This study investigated whether geldanamycin modulates TGFBI and C3 to reduce lymphocyte suppression. Metastatic pancreatic cancer cell lines (PANC 10.5, SW1990) and a pancreatic stellate cell (PSC) line were treated with geldanamycin, and their conditioned medium was used to treat peripheral blood mononuclear cells. Flow cytometry analysis was used to assess the effects on lymphocyte populations. We used independent T-tests to measure differences between the untreated and treated groups. Statistical significance was defined as a p-value < 0.05. The results showed that Th1 populations increased significantly in PANC 10.5-treated (p < 0.010) and PSC-treated (p < 0.019) groups, indicating enhanced anti-tumor immunity. Additionally, geldanamycin treatment led to reductions in tumor-promoting immune subsets, including Th2 in PANC 10.5-treated (p < 0.009), Th17 PANC 10.5-treated (p < 0.018), and Total T-cell in PANC 10.5-treated (p < 0.001) and SW1990-treated (P < 0.008) populations. Significant changes were observed in B-cells for PANC 10.5, SW1990, and PSC with p-values from 0.004–0.006, cytotoxic T-cells in PANC 10.5 (p < 0.032), and PSC-treated (p < 0.047). Total T-cells further highlight geldanamycin’s immunomodulatory potential. These findings suggest that geldanamycin may counteract pancreatic cancer-induced immunosuppression by restoring key lymphocyte subsets, providing a foundation for future therapeutic applications. This study underscores its potential as an adjunct immunomodulatory agent in metastatic pancreatic cancer treatment.