OAR@UM Collection:

Investigating acquired resistance in non-small cell lung cancer

Mon, 01 Jan 2024 00:00:00 GMT

Title: Investigating acquired resistance in non-small cell lung cancer Abstract: Introduction: Lung cancer is one of the leading causes of death in Malta, while 80% of all lung cancer cases are NSCLC. The EGFR is a crucial target in NSCLC as it signals several pathways of cell growth and proliferation. Third generation EGFR-TKI osimertinib inhibits the EGFR and in turn, inhibiting the growth of cancerous cells. Aim: To sensitize two adenocarcinoma cell lines, namely A549 (EGFR wild-type) and HCC827 (EGFR-mutant) by using osimertinib, a third generation EGFR-TKI. Methods: PrestoBlueÔ cell viability assays were carried out to evaluate the optimum concentrations of osimertinib on each cell line. Then, wound healing assays were carried out to assess the metastatic potential of each cell line treated with different concentrations of the third generation EGFR-TKI osimertinib. Results: A concentration-dependent decrease in percentage cell viability was observed in the A549 cell line. However, this was not the case with the EGFR-mutated HCC827 cell line, hence this cell line is resistant to osimertinib. The wound healing assays showed that osimertinib decreased the metastatic potential of the A549 cells. Conclusion: Osimertinib reduces proliferation and migration potential in the A549 adenocarcinoma cells but not in the HCC827 cell line, which reflects adenocarcinoma with an EGFR mutation. This research is a preliminary step to studying the effect of novel combinatory treatment with two ASOs for TCTP and HSP27. A combination which may possibly overcome the non-sensitivity seen by HCC827 to osimertinib, which may suggest resistance. Additionally, it can increase the effectiveness of osimertinib seen in A549 cells. Future work: The ALI model is mostly used to study NSCLC, especially when studying the pharmacological concepts. This 3D cell culture model mirrors the effects seen in vivo and the morphology and physiology of the cells are similar to what can be seen in vivo. These experiments are to be replicated in normal lung tissue cells and the cell cycle effects of the therapy are to be investigated using flow cytometry. Description: B.Sc. (Hons)(Melit.)

COPD-related molecular role of FBXO38 gene expression in epithelial airway cells

Mon, 01 Jan 2024 00:00:00 GMT

Title: COPD-related molecular role of FBXO38 gene expression in epithelial airway cells Abstract: Background: Chronic obstructive pulmonary disease (COPD) is a chronic and progressive inflammatory pathway disease which is, the third leading cause of death globally. It is mainly associated with smoking, however there are also genetic and environmental factors at play. Symptoms of the disease include breathing difficulties, excessive phlegm production, chesty coughing and, wheezing and airway structural changes. COPD is known to be a multifactorial and polygenic disease. When the right environmental and genetic factors interact, the risk of the disease increases significantly. FBXO38 has been identified as an expression quantitative trait locus (eQTL) and splicing quantitative trait locus (sQTL) target of SNV rs6889822. This SNV is a marker for COPD, which was found through GWAS studies. The FBXO38 gene is also a co-activator of KLF7, a transcription factor known to be involved in airway inflammation, however no studies have looked into the possible contribution of FBXO38 to COPD molecular pathways. Aim: The aim of this research is to study the effects of FBXO38 gene expression on COPDrelevant molecular pathways in human airway cells. Methods: H520, a high FBXO38 expressing epithelial airway cell line, was selected for the study, and the FBXO38 gene expression was knocked down using siRNA technology. Transfection parameters were first optimised using siGLO and after the ideal parameters were selected, cell samples were then transfected with FBXO38 mRNA-targeting siRNA, together with scrambled, non-transfected and untreated controls. RNA extraction was then carried out, and RNA-Sequencing (RNA-Seq) analysis was carried out on FBXO38 knockdowns and scrambled controls. Differential gene expression was carried out on the RNA-Seq data, followed by pathway analysis, in order to identify COPD-related molecular pathways that are modified by FBXO38 downregulation. Results: The results of the study showed that the knockdown of the FBXO38 gene resulted in the up regulation and down regulation of 25 genes with a significant q-value. KEGG pathway analysis identified 20 pathways which were enriched following FBXO38 knockdown of which nearly half are well known to be associated with COPD. GO analysis found two DEGs to be highly correlated with Transcription Factor AP-1 complex. Discussion: Four pathways from the KEGG analysis were shortlisted as being the most important due to being known to be highly related to COPD. These were the cAMP signalling pathway, interleukin-17 (IL-17) signalling pathway, Th1 and Th2 cell differentiation and finally, Th17 cell differentiation. Transcription factor complex AP-1 was identified from the GO cellular component analysis, being driven by two statistically significant DEGs. Conclusion: This project has determined that FBXO38 knockdown enriched several major COPDrelated pathways. This suggests that the gene relevance to COPD may be related to increased activity of these pathways. The results of this project add to current knowledge of molecular events in COPD, however more research is required to further enhance and solidify the data obtained. Description: B.Sc. (Hons)(Melit.)

Developing an analytical method for the determination and verification of cannabidiol (CBD) in recreational cannabis products against label claims

Mon, 01 Jan 2024 00:00:00 GMT

Title: Developing an analytical method for the determination and verification of cannabidiol (CBD) in recreational cannabis products against label claims Abstract: Following the recent legalisation of cannabis for recreational use in Malta, the market has seen a surge in the availability of cannabis-based products in shops. While the level of tetrahydrocannabidiol (THC) is not supposed to be higher than 0.2% in these products, the level of cannabidiol (CBD) is not determined in the legislation. Concerns have arisen regarding the quality and consistency of cannabis products available on the market. This burgeoning interest in these products has sparked extensive research into the accuracy of labelling and quantification of these cannabis products, especially the levels of THC and CBD. This present study aimed to address this gap by developing a validated method for the determination of the CBD content of commercially available cannabis products in Malta. A gas chromatography-mass spectrometry (GC/MS) technique was thus used for the development of a method for CBD quantification in such products. Methapyrilene HCl was used as an internal standard, and calibration standards were used to establish a calibration curve for the CBD. Samples of three different commercially available products, which were labelled as containing varying CBD concentrations (<14%, <17% and <6%) were purchased from a local shop. The three samples underwent rigorous preparation steps, including homogenisation, sonication, dilution with methanol, and filtration, in order to ensure that the samples were suitably prepared for eventual analysis by GC/MS. The results of the analysis revealed that there were notable discrepancies between the CBD concentration as stated in the respective label of the sample and the measured CBD concentrations in the sampled products. Specifically, Sample 1 exhibited a CBD content of 12.78% (below the value stated on the label of <14%), while Sample 2 displayed a significantly lower CBD content of 8.25% when compared to the stated value of <17%. Sample 3, however, demonstrated an average CBD percentage of 10.25%, exceeding the labelled value of <6%. Additionally, the Certificate of Analysis (CoA) also reflected differences in CBD content for Sample 3. The CoA for Sample 3 indicated a CBD content of 10.47%, which closely aligns with the measured value from this study (10.25%). However, this CoA value was higher than the <6% CBD content stated on the product label. This discrepancy highlights potential issues in the labelling process or batch-to-batch variability, suggesting that the actual product content may not consistently match the information provided to consumers. Overall, the study underscores the urgent need for improved regulation and standardisation in the commercial cannabis product industry, particularly in terms of accurate labelling and batch-to-batch consistency of CBD content. These findings have implications for consumer safety and highlight the necessity of regulatory oversight to ensure product quality and efficacy. Description: B.Sc.(Melit.)

An investigation of the use, effectiveness, and prescription of medical cannabis from a prescribers' perspective in Malta

Mon, 01 Jan 2024 00:00:00 GMT

Title: An investigation of the use, effectiveness, and prescription of medical cannabis from a prescribers' perspective in Malta Abstract: Background, Literature on the use and effectiveness of medical cannabis remains contradictory, despite its legalisation across many countries and states. Malta legalised the prescription of medical cannabis on the 18th of December 2018, by amending the Drug Dependence (Treatment not Imprisonment) Act, Cap 537. However, there remains uncertainty amongst Maltese prescribers on the use, effectiveness, and prescription of medical cannabis. Aims, The overall aim of this study was to gain a better insight into medical prescribers’ attitudes and beliefs, regarding the prescription of medical cannabis in Malta. Methods, In the first phase, an extensive scoping review was carried out to formulate the interview guide and the questionnaire with relevant questions regarding the use, effectiveness, and prescription of medical cannabis. In the second phase, a qualitative interview with a prescriber of medical cannabis in Malta was carried out. The interview guide was made of pre-determined open- ended and probing questions, based on the extensive literature review carried out and the tripartite structure of attitudes that was selected to be the study's theoretical framework. The initial objective of this study was to carry out qualitative interviews with registered active medical cannabis prescribers in Malta, but due to the exceptionally low engagement by registered cannabis prescribers, it was decided to reformulate the interview guide into an anonymous online questionnaire to be distributed to all potential prescribers in Malta. Therefore, in the third phase, the quantitative electronic questionnaire was posted to the Facebook group “Tobba Maltin” (Maltese Doctors) for the members to answer. Since, both a qualitative and a quantitative approach were chosen as data collection methods for this study, the study used a mixed-method approach. The questionnaire was based on the tripartite structure of attitudes framework, the literature review carried out for the interview guide, and some of the questions in the interview guide itself. The interview guide and the questionnaire were formulated to gain a better understanding of the prescription of medical cannabis in Malta, to identify the perceived effectiveness and safety concerns of medical cannabis amongst prescribers, and to gain insight into the regulatory approach involved in the prescription of medical cannabis. The results obtained from the two different data sets were first investigated separately and then together in the light of current research. Results, The interview and questionnaire findings were limited due to the low response rate but there was a decent representation of active medical cannabis prescribers in the questionnaire responses (31.25% response rate). The main findings were: - Prescribers decided not to prescribe medical cannabis because of a lack of confidence in their knowledge of medical cannabis, because its indications did not match their patients’ needs, and because of the limited evidence on medical cannabis effectiveness. - Most of the prescribers who prescribed medical cannabis did not have a high level of confidence when prescribing it. - Pain management was the most common indication for medical cannabis. - The use of international prescription guidelines has been shown to increase the level of confidence that prescribers have in prescribing medical cannabis. - Prescribers have a level of concern regarding the safety of medical cannabis, especially regarding dependency/addiction. - Local healthcare professionals do not possess the knowledge and skills to manage patients who are on medical cannabis. - There needs to be a balance between control and convenience regarding the regulatory process involved in prescribing medical cannabis. - Prescribers want more guidance when it comes to prescribing medical cannabis. - The prescription of medical cannabis has been affected by the legalisation of recreational cannabis. Conclusion, Findings indicate that no standard approach is being followed by Maltese prescribers regarding the prescription of medical cannabis. There is a need for interventions to teach and train Maltese prescribers how to manage patients on medical cannabis, promote local or international prescription guidelines, and remove the stigma associated with medical cannabis now that it has been legalised. The legalisation of recreational cannabis has further emphasised the need for national guidelines and policies guiding healthcare professionals on how to prescribe medical cannabis and how to manage patients on medical cannabis. Description: B.Sc.(Hons)(Melit.)