Theses (PHD)

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

To address gaps in the understanding of host-parasite interactions, this study employed a systems biology approach to investigate system biological responses in Balb/c mice infected with Toxoplasma gondii and Trypanosoma brucei brucei. The study found distinct histopathological changes in Balb/e mouse organs post- infection. Spleen hyperplasia, chronic inflammation, and immune cell infiltrations were apparent in both infected arms. Cytokine profiling identified key players like tumour necrosis factor alpha, interferon-gamma, interleukin-2 in T. gondii group interleukin-6, and interleukin-IO in T. b. brucei group, providing insights into their roles in host defence or immunomodulation. Nuclear magnetic resonance metabolomics profiling was used to identify potential parasitic infection biomarkers, revealing alterations in metabolic profiles indicative of metabolic adaptations and gut microbiota dynamics during infection. Notably, short-chain fatty acids like acetate and butyrate, aromatic amino acid derivatives like phenylacetylglycine and tyrosine, and metabolites such as sueeinate and creatine were implicated in immune regulation and host-parasite interactions. This study also explores interactions between the gut microbiota and parasitic pathogens, highlighting their impact on disease outcomes. Specifically, Lactobacillus species were identified as key players in modulating the host's immune response and metabolic processes during parasitic infections. In conclusion, the systems biology approach enhances our understanding of host-pathogen interactions in Balb,/c mice, revealing potential therapeutic targets. While the findings are specific to the mouse model, they offer valuable insights that could inform strategies for managing parasitic infections in humans. This study thus lays a strong foundation for future research aimed at developing targeted interventions across various host systems, potentially bridging the gap from animal models to human disease management.