Theses (PhD in Medical and Health Sciences)

Background: Bacterial biofilms within the root canal system are a major contributor in the failure of endodontic treatment, primarily attributed to the presence of significant pathogens such as Enterococcus faecalis (E. faecalis), Fusobacterium nucleatum (F. nucleatum), and Porphyromonas gingivalis (P. gingivalis), which demonstrate considerable virulence, antimicrobial resistance, and the capacity to penetrate dentinal tubules. Conventional irrigants such sodium hypochlorite (NaOCl) and chlorhexidine gluconate (CHX) have limitations including cytotoxicity, inadequate biofilm eradication, and adverse responses including tissue irritation and precipitate formation. Quaternary ammonium silane (codename- K21), a functionalised organosilicon compound with antibacterial and anti-inflammatory properties, not only disrupts microbial biofilms but also preserves compatibility with host tissue components, including macrophages. Comprehensive research is warranted to confirm K21's therapeutic benefits since it has potential to block Sortase Av(SrtA) and matrix metalloproteinases (MMPs), enzymes engaged in bacterial adherence and tissue breakdown. Objectives: The aim of this research was to assess K21's antibacterial and anti-inflammatory performance as an endodontic irrigant. Among specific objectives were comparing 1.00% K21 with 0.5% K21, 2.00% CHX, 6.00% NaOCl, and saline on single-species (E. faecalis, F. nucleatum and P. gingivalis respectively) and dual-species (F. nucleatum and P. gingivalis) biofilms at 200 μm and 400 μm dentinal tubule depths. Additionally evaluated in the study were K21's effects on hPDLSCs survival as well as RAW 264.7 macrophage viability and anti-inflammatory (M2) polarization. Moreover, molecular simulations were performed to assess the interaction of K21 with SrtA and MMP-9 molecular structures. While the null hypothesis predicted no significant differences, the hypotheses postulated that 1.00% K21 would show better biofilm reduction, higher macrophage and hPDLSCs viability, and greater anti-inflammatory benefits than conventional irrigants. Methods: This multimodal investigation utilized a combination of in vitro assays and imaging techniques. Biofilms of E. faecalis, F. nucleatum, and P. gingivalis were cultured on dentin specimens and exposed to different irrigants: saline, 6.00% NaOCl, 2.00% CHX, 0.5%K21, and 1.00% K21. Colony-forming unit (CFU) counts were used to assess bacterial viability, while structural changes were analyzed via scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Raman spectroscopy was employed to identify chemical alterations in post-treatment biofilm components. Cell viability was evaluated using the MTT assay and mitochondrial staining in RAW264.7 macrophages, while the alamar blue assay was utilised for hPDLSCs. Additionally, the expression of cytokines (TNF-α, IL-1β, TGF-β, VEGF-A) associated with macrophage polarization was evaluated using Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) and gene expression analysis to determine the immunomodulatory impact of all the irrigants. Molecular simulations were carried out inside the Schrödinger Drug Discovery Suite using the Desmond package. Molecular simulations analyzed K21’s binding to MMP-9 and SrtA molecular structures with stability assessed via root mean square deviation (RMSD) and binding free energy (MM-GBSA). Statistical analysis used ANOVA with Tukey’s post hoc test (p < 0.05) and Pearson’s correlation for depth-dependent efficacy. Results: The CFU analysis revealed that K21, especially at a 1.00% concentration, consistently showed enhanced antibacterial efficacy relative to 6.00% NaOCl and was frequently comparable to or more effective than 2.00% CHX against E. faecalis, F. nucleatum, P. gingivalis, and dual-species biofilms. For CFU findings of E. faecalis, at a depth of 200 μm and a duration of 1-minute, 1.00% K21 exhibited the lowest CFU at 1.358 ± 0.193, in contrast to 0.5% K21 (3.453 ± 0.205), 2.00% CHX (4.65 ± 0.093), 6.00% NaOCl (5.64 ± 0.108), and saline (9.63 ± 0.031), with statistically significant differences (p < 0.001). At 400 μm and 1-minute, similar trends were observed. In 5-minute exposures, 1.00% K21 consistently exhibited the lowest CFUs (0.9687–0.9924), significantly surpassing 2.00% CHX and 6.00% NaOCl at both 200 μm and 400 μm (p < 0.001). For F. nucleatum biofilm, 1.00% K21 exhibited the lowest CFUs (2.1199 ± 0.20274 at 200 μm/5 min and 2.1782 ± 0.55736 at 400 μm/5 min), demonstrating significant reductions compared to 6.00% NaOCl (p < 0.001) and comparable efficacy to 2.00% CHX. At 1-minute application, CFU counts were markedly diminished by K21 group compared to 6.00% NaOCl. For P. gingivalis, at 200 μm/1 min, 1.00% K21 (3.9831 ± 0.10972) and 0.5%K21 were significantly better than 6.00% NaOCl (5.1798 ± 0.39327) and were comparable to 2.00% CHX (4.0792 ± 0.10074). At 400 μm/1 min and 5 min, all test irrigants demonstrated effectiveness relative to saline; nevertheless, 1.00% K21 consistently exhibited the lowest CFU counts with no statistically significant difference from 2.00% CHX. In dual-species biofilm, 1.00% K21 demonstrated significant reductions compared to saline at both 200 μm and 400 μm for 1 and 5-minute treatments. At 200 μm/5 min, 1.00% K21 (4.6173 ± 0.37708) and 0.5%K21 (4.7104 ± 0.34656) demonstrated superior efficacy compared to 6.00% NaOCl (5.1432 ± 0.48289). 1.00% K21 was comparable to 2.00% CHX (p > 0.05) and significantly better than saline (p < 0.001) and 6.00% NaOCl (p = 0.046). Morphological analyses utilising SEM and TEM corroborated the CFU findings, demonstrating that 1.00% K21 induced significant membrane rupture, cytoplasmic leakage, and biofilm matrix disintegration across all the three species. The 1.00% K21 treated groups exhibited the most pronounced intensity deterioration, and Raman spectroscopy examination indicated notable spectrum shifts in the 480–490 cm⁻¹ range, so corroborating the hypothesis of heightened structural disruption of bacterial components. For macrophage viability, all groups showed cell viability above 80%. While 0.5% and 1.00% K21 showed higher viability (87.17% and 87.4%), viability reduced for 6.00% NaOCl (82.42%) and 2.00% CHX (86.08%). K21 groups were noticeably better than 6.00% NaOCl, although all test groups had lowered viability relative to saline (p<0.05). Fluorescence imaging depicted strong, homogeneous mitochondrial signals maintained by saline and 0.5% K21 that indicated healthy, viable cells. The 1.00% K21 group maintained general function while only modestly altered mitochondrial structure. While 6.00% NaOCl produced severe cytotoxicity, low cell density, and weak fluorescence, 2.00% CHX exhibited variable effects with some mitochondrial fragmentation. At a concentration of 0.5%, K21 demonstrated superior mitochondrial preservation compared to 2.00% CHX and 6.00% NaOCl. SEM and TEM analysis indicated that macrophages treated with saline exhibited normal integrity and morphology. K21 exhibited electron-dense inclusions at elevated concentrations, signifying stress, but preserving cell morphology with slight surface alterations. The 6.00% NaOCl treatment caused severe structural damage, membrane fragmentation, and cytoplasmic debris; 2.00% CHX treated cells showed surface shrinkage and internal aggregates. K21 group displayed generally better cellular preservation than CHX and NaOCl groups. Gene expression analysis demonstrated that K21 induced modest upregulation of pro-inflammatory cytokines (TNF-α: ~4.24–4.26; IL-1β: ~1.43–1.89), whereas 2.00% CHX and 6.00% NaOCl strongly upregulated both TNF-α (~19.38 and 19.30) and IL-1β (~2.30 and 1.89). Conversely, K21-treated groups exhibited significantly higher anti-inflammatory markers, with VEGF-A reaching 58.93 ± 0.16 for 1.00% K21 and TGF-β at 6.51 ± 0.11, substantially exceeding levels observed with CHX (TGF-β: 6.31 ± 0.66) and NaOCl. Overall, K21 demonstrated a balanced immunomodulatory profile characterized by reduced inflammatory activation and enhanced pro-healing capacity compared to conventional irrigants. At 30 minutes of exposure, the viability of human periodontal ligament stem cells (hPDLSCs) remained approximately close to 80% for all tested irrigating agents. Specifically, 1.00% K21 demonstrated a viability of 81.13%, 0.5% K21 at 80.13%, CHX at 80.16%, and NaOCl at 79.34%. Saline showed the highest cell viability at 98.48%. High viability was indicated by SEM and TEM studies of hPDLSCs showing intact morphology and membrane integrity from saline-treated cells. Severe structural damage, membrane lysis, and cytoplasmic disintegration were produced by 6.00% NaOCl. Moderate membrane distortion and vacuolation caused by 2.00% CHX indicated cytotoxic effects. Although 1.00% K21 showed some structural changes while 0.5%K21 maintained better integrity. TEM verified that although K21 caused subcellular changes, it maintained membrane continuity, hence indicating better biocompatibility than 2.00% CHX and 6.00% NaOCl. The K21 group shows a specific pocket binding on several MMPs and specific SrtA structures, according to molecular simulation, which generates a classic clouting effect. This can prevent SrtA enzymes and MMPs from acting catalytically, avoiding structural changes in dentin. Conclusions The 1.00% K21 group demonstrated superior antibacterial efficacy against E. faecalis, F. nucleatum, and P. gingivalis in dentinal tubules at 200 μm and 400 μm depths compared to 6.00% NaOCl and saline, with performance comparable to 0.5% K21 and 2.00% CHX. In dual-species biofilms, 1.00% K21 outperformed NaOCl and saline while showing equivalent results to CHX and 0.5% K21. It exhibited significantly enhanced biocompatibility and anti-inflammatory properties in RAW 264.7 macrophages and hPDLSCs compared to NaOCl, with results comparable to 0.5% K21 and CHX. K21 promoted M2 macrophage polarization and a balanced immune response. Molecular docking revealed specific binding affinity to MMPs and Sortase A, indicating a unique "clouting effect" that enhances antibacterial and regenerative capabilities. Overall, 1.00% K21 represents a promising biocompatible, multifunctional endodontic irrigant with superior antibacterial and anti-inflammatory potential. Keywords: antibacterial, human periodontal ligament stem cells, K21, macrophages, molecular simulation, quaternary ammonium silane.

Rheumatoid arthritis (RA) is a chronic autoimmune condition manifested by synovial inflammation and joint destruction. Tofacitinib was introduced for RA patients who have failed treatment with conventional synthetic or biologic disease-modifying antirheumatic drug (cs/bDMARDs). However, the interindividual variations in response may have both clinical and genetic aetiology. Hence, precise risk stratification based on clinical and genetic predictive markers may aid in identifying patients who can reap the full benefit from treatment management. This project aimed to explore the clinical and genetic factors associated with response to tofacitinib in RA patients. A retrospective medical data collection and the very first comprehensive whole genome sequencing was performed. Two third of the patients have responded to tofacitinib therapy. There was an increased odd ratio for baseline C-reactive peptide level, anti-citrullinated peptide autoantibody negativity, absence of bone erosion and baseline biologics naive associated with tofacitinib responders. Genomic analysis revealed that five significant variants with p value less than 1x10-7 were identified for the outcome measure of tofacitinib response. The strongest evidence for association to good response were with rs12607965_T variant of RNF125 gene followed by rs12149039_C variant of CDH13 gene, rs13315685_C of SLC12A8 gene, rs5749279_A of EIF4ENIF1 gene and rs13418335_C variant of GCC2-AS1 gene. The SEMA3F, SSH1, RNF138, SLC12A8, ADA2, ABCC13, ITGA8 and SORT1 genes were replicated from previous studies and related to RA inflammatory and destructive process. Furthermore, other genetic variants associated with tofacitinib responders were identified, which have not been reported previously i.e., CDH13, PCNX2, FEZ2, XRCC5, ZNF736, ARHGEF12, SLC12A8, EIF4ENIF1, GRID1, DMAC1, DRG1, GCC2-AS1, LIMS1 and RNF125. Overall, the identified predictive markers hold the potential to a broader precision medicine framework for RA, enabling more accurate treatment stratification and fostering cost-effective treatment allocation. Nevertheless, these markers needed further validation in larger and independent cohort prior to transforming into RA management that will reduce healthcare cost due to ineffective treatments, improving quality of life for patients and enhancing overall therapeutic efficacy.

Tocotrienols (T3) are a family of vitamin E that exists as four isomers: α-, β-, γ-, and δ-T3. γ-T3 and δ-T3 reportedly displayed promising anticancer potential in two-dimensional (2D) cell culture model and immunomodulating effects in animal model of various cancer types, especially breast cancer. However, there is a lack of clinical trials evidence supporting the use of tocotrienols for breast cancer patients. The disparity between preclinical and clinical results posed as a huge challenge for the clinical application of tocotrienols. Hence, with the use of in vitro three-dimensional (3D) tumour spheroid model of breast cancer, this study aimed to gain deeper insight into the anticancer potential of γ-T3 and δ-T3 in the presence of a complex tumour microenvironment. Tumour spheroid serves as a mimetic preclinical model due to their similarity with breast tumour characteristics. Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with limited therapeutic options. Two cell lines of different TNBC subtypes were employed: (1) MDA-MB-231, a mesenchymal-like subtype and (2) HCC38, a basal-like 1 subtype of TNBC. Tumour spheroid were successfully generated using Nunclon Sphera 96-Well U-Shaped-Bottom microplate and validated by morphology observation and gene expressions of 3D spheroids biomarkers using RT-qPCR, which were rigorously selected using data mining based on published studies of breast tumour spheroid. Notably, CD44, MMP1 and VEGFA were significantly upregulated in TS-MDA-MB-231 compared to 2D monolayer cells. Similarly, MMP1 and VEGFA were significantly upregulated in TS-HCC38. Cell proliferating assays (WST-1, CCK-8, and PrestoBlue HS) demonstrated that δ-T3 and γ-T3 were cytotoxic to the tumour spheroids and monolayer model derived from TNBC cells. The half-maximal growth inhibitory dose (GI50) of δ-T3 and γ-T3 in each model were determined. δ-T3 and γ-T3 were able to significantly and dose-dependently inhibit the viability of tumour spheroid in TS-MDA-MB-231 (δ-T3 27.44 μg/mL; γ-T3 19.44 μg/mL) and TS-HCC38 (δ-T3 38.21 μg/mL; γ-T3 19.44 μg/mL) , model albeit at a higher dosage compared to the monolayer model of MDA-MB-231 (δ-T3 11.53 μg/mL; γ-T3 12.01 μg/mL) and HCC38 (δ-T3 5.68 μg/mL; γ-T3 6.35 μg/mL). δ-T3 and γ-T3 induced ultrastructural changes in the tumour spheroid, including an increase in area, diameter and volume, as well as a decrease in circularity and solidity indicating cellular disaggregation. Total RNA of tocotrienols (δ-T3 or γ-T3) treated and untreated TS-MDA-MB-231 and TS-HCC38 were extracted with the Zymo Quick-RNA Miniprep Kit for transcriptome-level expression profiling using the Clariom D Affymetrix GeneChip platform. Tocotrienols (δ-T3 or γ-T3) regulated a total of 2567 and 5476 differentially expressed genes (DEGs) in δ-T3 and γ-T3 treated TS-MDA-MB-231, respectively. In TS-HCC38, 1004 and 278 DEGs were identified in response to δ-T3 and γ-T3 treatment, respectively, compared to untreated controls. In-depth functional bioinformatics analysis (Gene Ontology and KEGG pathway) of the transcriptomic profile revealed TNBC subtype-specific effects of tocotrienols in several pathways. Additionally, tocotrienols regulated a subset of long non-coding RNAs in TS-MDA-MB-231 which were further verified via comparison with the expression level of target mRNA. Notably, tocotrienol isoforms (δ-T3 or γ-T3) induced differential expression in pathways (oxidative phosphorylation, cell cycle, ribosome, proteasome), which were significantly downregulated in TS-HCC38, but an opposite upregulated effect of the same pathways was observed in TS-MDA-MB-231. Disease association analysis of the transcriptome profile revealed tocotrienol modulated clinically relevant pathways in breast cancer, δ-T3 significantly downregulated genes associated with cell cycle pathway, whereas γ-T3 downregulated genes associated with steroid hormone biosynthesis pathway in TS-HCC38. However, genes associated with cell cycle control and DNA damage response were upregulated in TS-MDA-MB-231. RT-qPCR validation demonstrated that both δ-T3 and γ-T3 differentially regulate MMP1, a key angiogenesis regulator and tumour spheroid biomarker, depending on the TNBC cell line used. Findings from this study suggest that tailoring tocotrienol treatment based on specific TNBC subtype is warranted to optimise therapeutic outcomes.

Microplastics and nanoplastics (MP/NPs) in the environment were found not only affecting environmental health but also impacting human health. Humans are exposed to MP/NPs via several routes including oral ingestion, inhalation, and direct skin contact. MP/NPs can penetrate biological barriers and reach different parts of human body. In the current study, the potential toxicity and pathways induced by polystyrene nanoplastics (PS-NPs) on normal human cells were evaluated. The viability of human cells was determined upon exposure to distinct concentrations and sizes of PS-MP/NPs. The toxicity of PS-MP/NPs was correlated with the particle size, concentrations, and exposure duration. The toxic effects of 20 nm PS-NPs treatment on human embryonic lung fibroblast (MRC5), immortalised human keratinocytes (HaCaT), and nasopharyngeal epithelial (NP69) cells were further investigated. Flow cytometric analysis showed that MRC5 and HaCaT cells experienced S phase arrest while NP69 cells halted at G0/G1 phase in the treatment groups. Apoptosis was seen in MRC5 and NP69 cells post-PS-NPs exposure. JC-1 and DCFHDA fluorescence assays revealed depolarisation of mitochondrial membrane potential (MMP) and oxidative stress in all treatment groups. PS-NPs exposure caused mitochondrial dysfunction in the exposed cell lines. Reactome pathway analysis of the transcriptomic data revealed adipogenesis and carcinogenesis may be affected in the treatment groups of MRC5, HaCaT and NP69 cells. Gene set enrichment analysis (GSEA) suggested the activation of nuclear factor kappa B (NF-B) of NP69 cells upon exposure to PSNPs and increased mRNA expressions of NF-B targeted genes were validated. In conclusion, PS-NPs induced oxidative stress, mitochondrial disorder, cell cycle arrest, and apoptosis, in MRC5, HaCaT, and NP69 cells, possibly impact on lipid metabolism and lead to cancer progression. Transcriptomic study revealed that PS-NPs induced mitochondrial dysfunction and endoplasmic reticulum (ER) stress partly through the activation of NF-B pathway, which triggers inflammatory responses via tumour necrosis factor (TNF) and interferon (IFN) signalling in NP69 cells. Identification of pathway and mechanism which control cellular toxicity induced by PS-NPs may enable pinpointing therapeutic targets in managing adverse health effects induced by PS-NP.

Background: The number of people in Malaysia who are 60 years of age or over has grown dramatically in the last several years, and by 2030, when this demographic accounts for more than 15% of the total population, the nation is predicted to become an ageing nation. Age-related changes in physiological processes result in a number of morbidities requiring more frequent visits to primary care and long-term care facilities and institutionalised care. Older people with various medical diseases are more likely to use multiple medications to manage their conditions, and the use of many medications increases the risk of exposure to potentially inappropriate medications. Older people who take multiple medications are at high risk of adverse drug reactions, and there is an increased risk of poor sleep quality in frail older people, including those living in aged care settings. Many drugs are known to alter sleep in older people; however, there is a lack of evidence on how potentially inappropriate medications are associated with sleep quality in aged care home residents in Malaysia. Therefore, this study evaluated the effects of potentially inappropriate medications, medication burden, and frailty on sleep quality and quality of life of aged care home residents in Malaysia. Methods: A total of 151 Malaysian aged care home residents were eligible and consented to the study. All of them were included in the data collection process, which was conducted in three phases: baseline, first follow-up in the sixth month, and second follow-up in the twelfth month. Data were collected using a comprehensive questionnaire comprising demographic data about the study participants and instruments to assess physical activity, mental health, frailty, quality of life, and sleep quality. The last part of the questionnaire involved gathering information such as comorbidities, medications, and physical health measurements. The mental health of the population was assessed using the hospital anxiety and depression scale (HADS), frailty was assessed using the Groningen Frailty Index (GFI), quality of life was assessed using the older of Life (OPQoL) scale, and sleep quality was assessed using Pittsburgh Sleep Quality Index (PSQI). All these scales are validated for use in the Malaysian-aged population. In addition to the above well-recognised tools, this study designed and validated a novel Medication Adherence Tool for older people in Malaysia, which was used in this study to assess the medication adherence of aged care home residents. The tool had good reliability and internal consistency for use in aged care home residents. Beers Criteria (2015) for potentially inappropriate medications, a Screening Tool for Older Person's Prescriptions (STOPP), and the Medication Appropriateness Index (MAI) were used to identify potentially inappropriate medications. A longitudinal study design was employed in which individuals were followed up three times a year (baseline and at six and twelve months). The data collected were analysed using SPSS version 28. Descriptive statistics were used to calculate percentage frequencies, mean and standard deviations. The differences in mean were calculated using an independent t-test. Correlation between study variables was assessed using Pearson and Spearman correlation methods. Multiple linear regression models were used to assess the association of continuous dependent variables with other study characteristics. Multiple logistic regression models were used to analyse the relationship between categorical variables. Results: 151 participants were recruited for the study, and 138 remained in the study at the one-year follow-up. The mean age of the study population was 74.45±8.43; 98% of the study population were Chinese, and 51% of the population were females. About one-third of the study population was exposed to potentially inappropriate medication according to both Beers Criteria and STOPP Criteria. Drugs with possible anticholinergic burden were present in nearly half (46%) of the older people in this study. There was an increase in polypharmacy from 29% at baseline to 34% at the second follow-up. The study developed a new Medication Adherence Tool (MAT) to assess medication adherence in aged care home residents. The tool had acceptable content validity and reliability (Cronbach's alpha = 0.890). The tool was found to be clear, simple, reliable, and free from ambiguity, making it suitable for use in the older adult population without requiring the expenditures associated with utilising current adherence assessment instruments. The study used an internationally recognised tool (PSQI) to assess sleep quality in aged care home residents and found that most of the study population has poor sleep quality. To the best of my knowledge, this is the first study assessing the effects of potentially inappropriate medications and other health-related factors on sleep quality. Frailty score (<0.001), anxiety (<0.001), depression (<0.001), and OPQoL (<0.001) significantly and independently predicted the sleep quality. However, potentially inappropriate medications and anticholinergic drug burden were not significantly associated with sleep quality. Age, gender, smoking, alcoholism, number of chronic conditions, polypharmacy, and medication inappropriateness cumulatively contribute to an increase in the prediction of global sleep quality scores even if they are independently not associated with sleep quality. A fixed effect regression analysis found that polypharmacy (p = 0.010, -0.24 at six months and p = 0.020 -0.22 at one year), OPQoL (p = 0.000 -0.54 at six months and p = 0.000 -0.53 at one year), and frailty (p = 0.009 0.18 at six months and p = 0.002 0.18 at one year) were significantly associated with changes in sleep quality scores. The combined model was significantly associated with a 23% increase in sleep quality score (R2 = 0.23, F (6,271) = 24.75, p < 0.001). The findings suggest that medication reviews should be conducted at regular intervals to reduce inappropriate polypharmacy, which could improve the sleep quality of the aged care home residents. The study assessed the effects of sleep quality, frailty, and medication-related factors on the quality of life in older adults. Frailty, anxiety, depression, and sleep quality were independently and significantly associated with quality of life. A stepwise regression analysis was used to identify the strongest predictors of older people's quality of life. Global PSQI (p = 0.001 -0.28 at six months and p <0.001 -0.36 at one year) and depression (p = 0.006 -0.23 at six months and p <0.001 = -0.30 at one year) were most significantly associated with quality of life. Since sleep quality in the aged care home residents was the strongest predictor of quality of life, improving sleep quality could positively impact residents' general health and well-being. In addition to sleep quality and quality of life, the study assessed the factors influencing frailty in aged care home residents. Frailty was associated significantly with PIM (OR = 4.22, 95%CI [1.80-9.88]), STOPP criteria (OR = 2.95, 95% CI [1.426.16]), MAI (OR = 1.17, 95%CI [1.05-1.30]), anticholinergic burden (OR = 2.36, 95%CI [1.26-4.43]), and DBI (OR:3.92, 95%CI [1.43-10.76]) only at the first followup. More long-term studies are required to determine the effect of medication inappropriateness on frailty, as the other phases of the study could not find any significant association. However, frailty was significantly associated with anxiety (OR = 1.49, 95%CI [1.25-1.78], and OR = 1.72, 95%CI [1.38-2.15] respectively), depression (OR = 1.58, 95%CI [1.31-1.90] and OR = 1.57, 95%CI [1.29-1.90] respectively) and sleep quality (OR = 1.28, 95%CI [1.08-1.52] and OR = 1.34, 95%CI [1.13-1.59] respectively) in the first and second follow-ups (p <0.01in all cases). The most significant association was found between frailty and OPQoL scores, where the association was consistent in all three phases of the study (OR = 0.92, 95%CI [0.86-0.98], OR = 0.93, 95%CI [0.89-0.98] and OR = 0.96 95%CI [0.920.99] at baseline, six months and 12 months, respectively). Preliminary findings from this PhD study were published in PLOS One and MDPI Pharmacy. Other findings will be submitted for publication after the thesis has been submitted for examination to ensure that examination deadlines for this part-time thesis are met. Conclusion: This PhD study used a unique combination of internationally recognised instruments to determine the impact of medication inappropriateness, burden, and frailty on sleep quality and quality of life among older people in aged care homes. The novel Medication Adherence Tool was validated and used to assess the medication adherence of aged care home residents in Malaysia. The tool is simple, easy to use, less time-consuming and more economical compared to many existing medication adherence tools for use in the older population. Most of the study population had poor sleep quality, and the prevalence of potentially inappropriate medications was high enough to cause some concern. Even though there was no significant association between sleep quality, medication appropriateness parameters, and anticholinergic burden, the study suggests medication reviews to reduce drugrelated problems and improve health outcomes. Sleep quality was significantly associated with quality of life, polypharmacy, and frailty. These findings further support the need for medication reviews and highlight the need to assess the quality of life and frailty when conducting the reviews and/or designing interventions to reduce inappropriate polypharmacy. In addition, the study found a bidirectional relationship between sleep quality, quality of life, and frailty. Although frailty is nonmodifiable, this study highlights that adjusting the modifiable factors (medication appropriateness and medication burden) can potentially improve sleep quality and quality of life. Paying particular attention to addressing frailty and improving modifiable factors in older people residing in aged care homes could improve health outcomes and reduce healthcare costs to individuals and society. The generalisability of the present study to Malaysian aged care homes is restricted due to the significant proportion of the Chinese population in the study. A similar longitudinal study over a longer period in a larger population, which includes a cost-benefit component, would provide further insight into factors influencing sleep quality and quality of life. Keywords: Sleep quality, Potentially Inappropriate Medications, Older People's Quality of Life, Frailty, Aged care homes