/library/oar/handle/123456789/117811 2025-12-26T09:05:19Z /library/oar/handle/123456789/125721 Title: Investigating the cytotoxic effects of AZD5363 on lung cancer cells Abstract: Background: Lung cancer is reported to be the most commonly occurring cancer in the world, being the leading cause of cancer death among men, and the second most common cause of death among women after breast cancer (Sung, Ferlay et al. 2021). The PI3K/Akt/mTOR pathway has been strongly linked to the development and progression of NSCLC, which constitutes 85% of all lung cancers (Cheng, Shcherba et al. 2014, Papadimitrakopoulou 2012). This pathway is therefore a compelling target for anticancer therapy in NSCLC due to the extensive downstream signalling effects it has on the onset and progression of malignancy, as well as its potential impact on response and resistance to conventional therapies (Tan 2020). Among the PI3K/Akt/mTOR inhibitors is AZD5363 – a very potent and orally bioavailable pyrrolopyrimidine-derived pan-Akt kinase inhibitor which competes with ATP for Akt kinase association at the ATP binding site (Andrikopoulou, Chatzinikolaou et al. 2022). Besides the ongoing National Lung Matrix Trial (Middleton, Fletcher et al. 2020), few other investigations have, to date, tested the efficacy of this drug in different types of lung tumours. Aim: This study therefore aimed to investigate AZD5363 by sensitising two forms of aggressive NSCLC cells. Methodology: PrestoBlueTM cell viability assays were carried out to test the cytotoxicity of varying concentrations of AZD5363 on H520 (derived from squamous cell carcinoma) and A549 (originating from adenocarcinoma) cell lines. With the identified concentration that resulted in a 30% loss in cell viability, an ELISA assay was performed on A549 cells, to quantify how much of the protein eIF4E was being decreased. The same drug concentration was also utilised for wound healing assays, to investigate cell migratory behaviour of A549 cells in response to drug treatment. Results: The treatment of 10 – 100 μM AZD5363 on H520 cells failed to achieve a 30% loss in cell viability, and therefore no further investigations were conducted on this cell line. With respect to A549 cells, the same treatment decreased cell viability in a concentration-dependent fashion, such that 55 μM AZD5363 was selected as the 30% inhibitory concentration (IC30) for the subsequent investigations carried out on this cell line. In the ELISA assay, treatment of A549 cells with 55 μM AZD5363 achieved a reduction of 29% in mean eIF4E concentration. Following wound healing assays, percentage wound closure of A549 cells treated with 55 μM AZD5363 reached 33% after 72 hours of treatment, compared to 11% reached by cells treated with vehicle control solution. Conclusion: The reduction in mean eIF4E concentration achieved with 55 μM AZD5363 (IC30), as well as the migratory inhibition observed, indicate that this drug may be further investigated in combination with chemotherapy or other investigational treatment regimens, to potentially improve survival and quality of life. Description: M.Sc.(Melit.) 2023-01-01T00:00:00Z /library/oar/handle/123456789/125637 Title: Poisoning related admissions to the Intensive Therapy Unit at the general teaching hospital in Malta Abstract: Background: Studies worldwide have shown a trend toward an increase in number of Intensive Therapy Unit (ITU) admissions due to intentional and unintentional poisoning. However, there is a paucity of publications and knowledge about the characteristics and demographics of patients admitted to local ITUs for the management of such conditions. The aim of this study was to conduct a fully anonymised retrospective analysis of patients admitted to the ITU at the Mater Dei Hospital, the general teaching hospital in Malta with poisoning related issues and to identify emerging admission patterns and assess the progress and outcome of such patients. Methods: A detailed fully anonymised retrospective observational study from anonymised patient records was conducted following ethics and data protections approval. The data of all patients admitted to the ITU over 18 years of age for any poisoning related issues from the 1st of January 2015 to the 31st of December 2021 were analysed. Medical information collected included the nature of drug toxicity or overdose, including reason for admission to ITU, management in ITU, length of stay in ITU and outcome of patient. The data was analysed using the statistical package for social sciences program (SPSS). Results: In the period from January 1, 2015, to December 31, 2021, there were a total of 3,394 adult patient admissions to the Intensive Care Unit (ITU) at Mater Dei Hospital. Among these admissions, a total of 252 were related to poisoning, and thus included in this study. The age of patients ranged from 18 to 87 years, with a median age of 39.5 years (Interquartile Range [IQR]: 28 to 55 years). Gender distribution showed 94 females (37.3%) and 158 males (62.7%). The study identified seven most frequently involved causes of poisonings, namely recreational drugs(19.9%), sedatives (13.9%), antidepressants (11.3%), antipsychotics (11.3%), alcohol (10.7%), analgesics (6.9%) and antihypertensives (4.6%). Patient survival rate was 97.6%, and the mortality rate was 2.4%. Among the findings, it was observed that elderly patients over 60 years old with comorbidities experienced a longer duration of stay in the ITU compared to younger patients. Conclusion: This study, being the first of its kind in Malta, provides key insights into the critical issue of poisoning-related admissions to the intensive therapy unit in Malta. By investigating the diverse factors contributing to these admissions, such as the demographics, common substances involved in overdose, Glasgow Coma Scale score at time of hospital admission, intent of overdose, management of poisoning patients in ITU, patient outcome, and length of stay in ITU, this study offers a thorough understanding of the various complexities involved. Moreover, this research highlights the necessity for further study in the field of toxicology in order to ensure better patient care and patient outcomes. Description: M.Sc.(Melit.) 2023-01-01T00:00:00Z /library/oar/handle/123456789/125626 Title: An in vitro model to measure Nerium oleander toxicity using Hep G2 cell line Abstract: Introduction: Nerium oleander is an evergreen shrub belonging to the Apocynaceae family which is locally found as an ornamental shrub decorating roadsides and gardens. All parts of oleander contain cardiac glycosides such as oleandrin, folineriin, adynerin and digitoxigenin which are potentially fatal when ingested. Various factors influence the glycoside content including the season, location, part of the plant, and the colour of the flower. Cardiac glycosides adversely affect multiple organ systems, with the cardiovascular and gastrointestinal system being the most studied, however other organs including the liver are also adversely affected. This study aimed at developing an in vitro model to test the toxicity of cardiac glycosides extracted from local species of Nerium oleander on hepatic cells. Methods: Leaves and twigs belonging to white flowered and pink double flowered cultivars were collected during summer, dried, and extracted with ethanol using the Soxhlet apparatus. The toxicity of these extracts was evaluated against human hepatic tumour cells (Hep G2) via MTT colorimetric assays using concentrations ranging from 0.05 ppm to 5 ppm over a duration of 24, 48 and 72 hours.Results: Data indicates that there is a positive correlation between concentration of the extract and cell death for all concentrations of pink double flower extracts tested, likely via apoptosis. In case of white flower extracts, results were varied. Time dependent toxicity was observed for pink double flower extracts, but not for white flowered extracts. A direct comparison in cell viability between pink double flowered extracts and white flowered extracts revealed a significant difference between leaves extract, but not for the twigs extract. Generation of IC50 values demonstrated that the pink double flower leaves and twigs extracts exhibited the highest toxicity at a duration of 48 hours, where a concentration of 0.03 ppm was modelled to be fatal to 50% of the cells. Conclusion: This study established an in vitro model for assessing toxicity of Nerium oleander extracts on hepatic cells using the Hep G2 cell line, which demonstrated that the pink double flower extracts demonstrated higher toxicity than the white flower extracts, likely attributed to varying cardiac glycoside levels. This highlights the importance in local plant species variations, and the possibility of using flower coloration as an indicator for cardiac glycoside content and toxicity in Nerium oleander. Description: M.Sc.(Melit.) 2023-01-01T00:00:00Z /library/oar/handle/123456789/119432 Title: Gene regulation and small molecule therapeutics in non-small cell lung cancer Abstract: Background: Lung cancer is one of the commonest cancers worldwide and the highest cause of cancer related mortality. Mutations in epidermal growth factor receptor (EGFR) affect the mTOR pathway, which is essential for protein translation and which is frequently dysregulated in non-small cell lung cancer (NSCLC). Aberrant EGFR signalling confers resistance to chemotherapy, making it difficult to treat. Several EGFR tyrosine kinase inhibitors (TKIs) are available but although these are initially effective, resistance is inevitable. Afatinib is a 2nd generation TKI indicated to treat EGFR mutant NSCLC but is subject to resistance by emergence of new mutations. Translationally controlled tumour protein (TCTP) is a protein which affects mTOR signalling. Inhibiting the mTOR pathway via EGFR using afatinib and via TCTP using an antisense oligonucleotide (ASO) simultaneously might be useful in treating EGFR mutant NSCLC. Aims: The aim of this project is to investigate a novel combinatory regimen of EGFR TKIs and TCTP ASO vs EGFR TKI or TCTP ASO monotherapy on EGFR mutant NSCLC models. Methods: HCC827 and H1975 cell lines were treated with afatinib and transfected with TCTP ASO alone or in combination and their effects were measured using viability assays and wound healing assays. Enzyme linked immunosorbent assay (ELISA) was employed to measure eIF4E protein, downstream of mTOR as an indication of afatinib effect. Real time quantitative polymerase chain reaction (RT-qPCR) was used to confirm knockdown of TPT1 gene by TCTP ASO. Results: Both afatinib and TCTP ASO were effective in reducing cell viability in both cell lines. Further, afatinib affected cell migration in both cell lines whereas TCTP ASO only had an effect on H1975 cell migration. Combinatory therapy with afatinib and TCTP ASO was more effective in reducing cell viability in both cell lines than afatinib or TCTP ASO monotherapy. Description: M.Sc.(Melit.) 2023-01-01T00:00:00Z