Saudi Cultural Missions Theses & Dissertations
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Item Restricted Assessment of Immunotherapy-Related Adverse Events Among Patients with Cancer in Saudi Arabia: A Mixed Methods Study(Queensland university of techonology, 2024) Alquzi, Fatimah; Bradford, NatalieThis thesis contributes significantly to cancer care by focusing on patients undergoing immunotherapy in Saudi Arabia. it is the first study to explore the challenges these patients face, offering valuable insight into their experience. the research translated and validated an immunotherapy assessment tool and assessed its real world use by evaluating its acceptability, feasibility, and clinical utility. this study deepens the understanding of immune related adverse events and provide critical perspectives for clinicians, patients, and researcher. Ultimately, it contributes to improving the management of adverse events in cancer immunotherapy. Background: Cancer is a rapidly growing healthcare system challenge, with more than 19.3 million incidences recorded globally in 2020; this is projected to surpass 29.5 million by 2040. The emergence of immunotherapy has positively changed many advanced cancer outcomes and improved survival rates. Despite immunotherapy being an efficacious and reliable treatment option for cancer, its success is tempered by associated immune-related adverse events (irAEs). Limited understanding of proactive irAE assessment further complicates this issue. Patient Reported Outcome (PRO) measures are standardised tools used to systematically collect data from patients about their health status, symptoms, functioning, and well-being, and are increasingly advocated for use in routine clinical care. However, their use in clinical care, and more specifically to assess irAEs and symptoms, is an understudied area. Objectives: To investigate the feasibility, acceptability, and usefulness of systematic assessment of irAEs from the perspectives of patients with cancer living in Saudi Arabia. Methods: The doctorate project involved five inter-related studies, underpinned by the theory of Symptoms Experience in Time (SET), a conceptual framework aimed at understanding the temporal dynamics of symptom manifestation and progression. In Study 1, a systematic literature review was completed. A comprehensive search strategy was employed to identify relevant literature on symptom assessment and management strategies related to irAEs in cancer patients. The review identified a PRO tool developed specifically for irAEs to assess 20 symptom and six interference items. In Study 2, the PRO tool identified for irAEs assessment from the review underwent robust translation from English into Arabic using the Brislin translation method. This involved forward and backward translations, consensus committees with health experts and linguists, and iterative adjustments to ensure linguistic accuracy and cultural appropriateness. Study 3 was an observational study undertaken in Saudi Arabia to evaluate the psychometric properties of the tool among Saudi Arabian cancer patients undergoing immunotherapy. The assessment involved mixed methods, including cognitive interviewing to assess item clarity and relevance, content validity and test–retest reliability analysis, and a post-assessment feedback survey to assess acceptability. Study 4 was a prospective observational cohort study over four weeks that investigated the feasibility, acceptability, and usefulness of the translated tool to assess irAEs and symptom experiences of patients undergoing immunotherapy for cancer in Saudi Arabia. Finally, in Study 5, a subset of participants from Study 4 participated in in-depth semi-structured interviews, which were analysed using qualitative content analysis. Results: Study 1: The systematic literature review identified a suitable PRO tool for assessing diverse irAEs among cancer patients undergoing immunotherapy. The review also identified gaps in evidence, including the unclear frequency of symptom assessment and the role of non-pharmacological approaches for managing mild irAEs. In Study 2, the PRO tool was translated, creating the Arabic version of the tool: the Arabic Immunotherapy Symptom Assessment Inventory (AISAI). Study 3 evaluated the psychometric properties of the AISAI, which demonstrated content validity, reliability, linguistic precision, and cultural relevance. The Cronbach’s alpha coefficients for the internal consistency were calculated as 0.90 for the 20 symptom items and 0.88 for the interference scale, indicating satisfactory reliability. The test–retest reliability, assessed through intraclass correlation coefficients (ICC), showed excellent agreement between Time 1 and Time 2, with ICC values above 0.90. In Study 4, a real-world, 4-week evaluation involving a cohort of 69 patients undergoing cancer immunotherapy treatment was completed. The feasibility and acceptability of administering the AISAI were high, with 97.1% affirming its acceptability and applicability. The five most prevalent and severe symptoms, based on mean scores, were: numbness or tingling; pain; rash or skin change; interference with general activity; and impaired walking. Over the 4- week immunotherapy period, there was a notable increase in the severity of symptoms, with statistically significant changes observed. The number of participants with moderate (score 4–6) and severe (score 6–10) symptoms increased, indicating a worsening pattern over time. In Study 5, a qualitative evaluation affirmed that the AISAI was perceived as a valuable tool for early recognition and assessment of irAEs, with a preference for routine usage. The evaluation also highlighted knowledge gaps, emphasising the need for educational interventions to enhance comprehension and management capabilities among patients. Conclusion: This thesis represents a novel contribution to the field, particularly in the context of cancer patients undergoing immunotherapy treatment in Saudi Arabia. It introduces an innovative approach by incorporating PROs in Saudi Arabia. This marks a significant advancement in the assessment practices related to irAEs. Notably, it is the first study to explore the experience of irAEs in Saudi Arabia using PRO, providing a comprehensive understanding of the challenges faced by these patients. Moreover, this research pioneers the investigation of the use of the AISAI in real-world settings for cancer patients undergoing immunotherapy. It assesses the tool’s acceptability, feasibility, and usefulness, shedding light on its practical implications in the clinical setting. The research contributes a valid and comprehensive tool, the AISAI, designed specifically for assessing irAEs in Arabic cancer patients. This research makes a valuable contribution to clinicians, patients, and researchers, enhancing the overall understanding and approach to assessment of adverse events in the context of cancer immunotherapy.13 0Item Restricted Subcellular Location-Dependent Regulation of Interferon-Induced Transmembrane Protein 1 in Glioblastoma(The University of Edinburgh, 2024) Mubarak, Rawan; Ball, KathrynGlioblastoma multiforme (GBM) is one of the most aggressive and lethal brain cancers, known for its highly invasive nature and resistance to conventional therapies. Central to this resistance is the presence of glioblastoma stem cells (GSCs), which contribute to tumour recurrence and heterogeneity. This thesis investigates the roles of interferon-induced transmembrane proteins (IFITMs), specifically IFITM1 and IFITM3, within GSCs and their potential as therapeutic targets. The study provides a detailed analysis of IFITM1’s expression, subcellular localisation, and interaction with other proteins in response to interferon stimulation, employing advanced molecular techniques such as co-immunoprecipitation, immunofluorescence microscopy, and proteomic analysis. Key findings include the discovery that IFITM3 plays a critical role in regulating IFITM1’s expression and localisation, with significant implications for IFITM1’s function in cancer cell biology. This thesis also contributes to validating a novel interaction between IFITM1 and Lysosomal-associated membrane protein 1, suggesting a potential role for IFITM1 in autophagy, which could be pivotal in GBM’s resistance to treatment. These insights not only advance the understanding of IFITM proteins in GSCs but also highlight their potential as targets for therapeutic intervention in GBM. This work lays the foundation for future studies aimed at manipulating IFITM proteins to develop novel strategies for overcoming GBM treatment resistance.38 0Item Restricted Role of somatic copy number alterations in the aetiology and prognosis of Acute Myeloid Leukaemia(Newcastle University, 2024-02) Alharbi, Abrar Abdulghani; James, AllanAcute Myeloid Leukaemia (AML) is a group of highly heterogeneous haematological malignancies that arises from the accumulation of acquired genetic lesions, including point mutations, translocations, and DNA copy number alterations (CNAs) that confer a survival advantage to the leukaemic cell. The identification and characterisation of these aberrations have not only shed light on the pathogenesis of AML but also have improved patient stratification and the selection of therapeutic strategies. Despite these advances, drug resistance and relapse remain persistent clinical challenges, which may reflect the presence of as yet undetected alterations that influence disease progression and outcome. Recent advances in high throughput platforms such as microarray‐based genotyping technologies have revealed previously unrecognised cryptic chromosomal aberrations associated with DNA copy number changes. Given the heterogeneity of AML genomes, input from these platforms can potentially supplement cytogenetic data to improve prognostication and guide treatment decisions, and perhaps lend further insight into the mechanisms underlying AML pathogenesis. In this study, we analysed SNP‐microarray data from a large cohort of over 3000 AML cases for CNAs and copy‐neutral regions of homozygosity. In addition to providing a comprehensive overview of the landscape of acquired CNAs and regions of homozygosity in AML, this large cohort enabled us to identify and validate recurrent novel focal CNAs. Among validated gene targets affected by focal CNA is MIR4447, a previously uncharacterised microRNA. To investigate the potential role of MIR4447 in AML pathogenesis, we developed both gain‐ and loss‐of‐function cell models via gene overexpression and CRISPR/cas9‐mediated gene editing, respectively. These models were interrogated using RNA sequencing to analyse the transcriptional impact of miR‐4447 modulation. Our findings suggest that miR‐4447 has a multifaceted and context‐dependant role in regulating cellular functions, impacting energy metabolism, ribosome synthesis, immune response, and possibly leukaemogenesis through alterations in nutrient transport, mitochondrial function, and cell migration. Collectively, our findings contribute to our understanding of the genomic intricacies of AML and highlight the need for further research to investigate the downstream targets of miR‐4447, its effects in different cellular contexts, and its interaction with other regulatory molecules.7 0Item Restricted Field Cancerization and Microbiome Effects on Lung Cancer: A Source of Early Detection Biomarkers to Improve Patients’ Outcome(George Mason University, 2024-08-16) Alhammad, Rayan; Luchini, AlessandraLung cancer results in more deaths than any other cancer in the United States and worldwide, with non-small cell lung cancer (NSCLC) accounting for most cases. Diagnosis typically involves chest imaging, molecular testing, and biopsy. However, most patients are diagnosed at advanced stages, with only a 6% chance of a 5-year survival rate. In contrast, early-stage diagnosis and treatment can result in a favorable prognosis, with a high 5-year survival rate of 70-90%. The concept of tumor field cancerization describes a phenomenon where exposure to carcinogens can cause histologic changes in large areas of tissue, creating a field of pre-malignant cells that can eventually develop into tumors. Additionally, microbiota dysbiosis might influence tumor development. Studies have identified several commensal bacteria present in the lower airway tracts, such as Streptococcus, Prevotella, and Veillonella. The high mortality rate of lung cancer is often attributed to i) its late-stage diagnosis, ii) aggressive nature given its ability to metastasize early in the disease process complicating treatment and reducing survival rates, and iii) significant therapeutic challenges despite current treatments such as surgery, chemotherapy, radiation therapy, targeted therapy and immunotherapy. Despite advancements, the survival rate for advanced lung cancer remains low. To address this challenge, our research focuses on identifying risk protein biomarkers that are associated with the earliest molecular changes indicative of an ongoing tumorigenic process, thus offering significant potential for early intervention. Our study investigates the phenotypic molecular changes in the bronchial tree of NSCLC patients in light of the field cancerization theory and correlates these findings with blood biomarkers to support the future development of a non-invasive risk assessment test. Using enhanced liquid chromatography tandem mass spectrometry (LC-MS/MS) proteomic analysis and two independent cohorts of lung cancer patients (N=18, and N=263) with matched plasma and bronchial tree tissue specimens, we identified a set of 6 and 13 candidate risk plasma biomarkers with tissue origin. Additionally, we explored the microbiome proteome composition in NSCLC patient tissue and plasma to support future characterization of its potential role in cancer development. Risk biomarkers will enable the evaluation of individuals at high risk, guiding necessary lifestyle adjustments and facilitating the development of personalized prevention plans and therapies.48 0Item Restricted Advancing Oncolytic Virotherapy: The Role of Chemical Sensitizers in Enhancing Viral Oncolysis in Resistant Cancers(University of Ottawa, 2024-07-28) Alwithenani, Akram; Diallo, Jean-SimonOncolytic virotherapy, leveraging viruses to target cancer selectively, has shown promise with the FDA-approved oncolytic herpes simplex virus-1 (HSV-1) for melanoma treatment. However, the efficacy of oncolytic viruses (OVs) varies across cancer types, highlighting the need for strategies to sensitize resistant tumors. This thesis investigates the potential of Dimethyl fumarate (DMF), its analog Tepilamide Fumarate (TPF), and novel synthetic small molecules identified from a high-throughput screen to enhance OV effectiveness in cancer therapy. DMF, approved for multiple sclerosis and psoriasis, and TPF, under trial for psoriasis, were evaluated for their ability to boost HSV-1 and vesicular stomatitis virus (VSVΔ51) activity against cancer cells. Our findings reveal that pre-treatment with DMF or TPF significantly increases HSV-1 and VSVΔ51 replication in various cancer cell lines, including melanoma, and improves viral oncolysis. Notably, both DMF and TPF enhance OV infection in mouse-derived tumor cores and human tumor samples, while TPF exhibits a remarkable capacity to heighten VSVΔ51 infection and cell killing, outperforming DMF in vitro. Both compounds achieve these effects by downregulating the interferon (IFN) pathway, rendering cancer cells more susceptible to viral infection. Additionally, we demonstrate the ability of DMF and TPF to boost gene therapy vectors' transduction efficiency, which points to the broader utility of these drugs in gene therapy. Further exploration through a high-throughput screen identified several small molecules that sensitize human renal carcinoma cells to HSV-1 and VSVΔ51, highlighting potential new avenues for overcoming tumor resistance to OVs. These compounds enhance viral replication and oncolysis, presenting a promising path for future oncolytic virotherapy research and development. The synergistic potential of combining approved therapies like DMF with OVs, the promising effects of TPF, and newly identified small molecule sensitizers underscore the feasibility of enhancing OV efficacy in resistant cancers. This study not only broadens our understanding of how small molecules can potentiate oncolytic virotherapy but also sets the stage for clinical evaluation and the development of more effective, personalized cancer treatment strategies. Collectively, these findings advocate for further investigation into DMF, TPF, and other sensitizing compounds to unlock their full therapeutic potential in oncolytic virotherapy.14 0Item Restricted Rme-6, a Novel Regulator for EGFR Trafficking and Signaling(University of Sheffield, 2024-02-21) Alshahrani, Fahad Ayidh M; Smythe, ElizabethThe Epidermal Growth Factor Receptor (EGFR) is a key regulator for critical cellular processes including proliferation, migration, and apoptosis. Dysregulation of EGFR trafficking and signaling plays a crucial role in cancer development and contributes significantly to resistance to chemotherapy. This thesis examines the novel regulatory role of Rme-6, a Rab5 guanine nucleotide exchange factor (GEF), in modulating EGFR signaling by influencing its endocytic flux, a key determinant in the spatial and temporal dynamics of EGFR-mediated signaling pathways. Through a series of molecular and cellular experiments, I demonstrate that Rme-6 critically influences EGFR endocytic trafficking. Specifically, loss of Rme-6 results in increased accumulation of EGFR in APPL1-positive endosomes. This accumulation alters the downstream signaling fate of ERK1/2 by modulating its nuclear translocation. I have shown that Rme-6 has a positive effect on ERK1/2 signaling, while its disruption leads to aberrant ERK1/2 activity. This change in ERK1/2 signaling correlates with altered cell proliferation rates, suggesting a potential mechanism for cancer progression. Additionally, I reveal that Rme-6 may act as a scaffold for CK2 to phosphorylate ERK1/2 on its SPS motif, which is essential for its nuclear translocation and activation of transcription factors such as c-Fos. This phosphorylation impacts gene expression and subsequent cell fate decisions. This research not only advances our understanding of the molecular dynamics of EGFR signaling but also proposes Rme-6 as a potential therapeutic target in cancers characterized by dysregulated EGFR signaling pathways.16 0Item Restricted Orofacial Development Changes in Children Following Cancer Treatment: A Comprehensive Review of Literature and Analysis of Current Data in Leeds Dental Institute.(University of Leeds, 2024) Alghamdi, Talal; Drummond, BernadetteIntroduction: Childhood cancer survivors often experience various side effects after treatment, including dental and orofacial developmental conditions. According to the literature, the treatment for cancer in children can affect the development of teeth, the function of salivary glands, the development of facial structures, and the operation of the temporomandibular joint [TMJ]. Leeds Dental Institute [LDI] has accumulated a wealth of data while providing dental healthcare for cancer survivors. This extensive data has not been thoroughly explored or published. Thus, this study aims to investigate the long-term effects of cancer treatment on dental and orofacial structures from the literature and the available records in children at Leeds Dental Institute. Methods: This research is structured into two sections. The first is a comprehensive literature review of existing studies on the adverse effects of cancer treatments on oral and facial structures in children by searching six databases to establish a foundation for understanding the broader context of the issue. The second section is a retrospective data collection and analysis of paediatric patient data from the electronic records in LDI using a list of appointments attended by cancer patients in LDI. Results: Fifty-one articles were included in the comprehensive literature review following the database search and the inclusion criteria. Numerous studies concluded that chemotherapy and other anticancer treatments in children are linked to increased dental anomalies like microdontia and enamel defects, especially when treatment occurs at a young age. The findings have been summarised in tables. Of the 806 registered appointments identified, the clinical records of 85 childhood cancer survivors who met the inclusion criteria were included. The post-treatment identified conditions included microdontia, hypodontia and enamel hypoplasia. Demographics, cancer diagnosis and type of treatment, in addition to dental findings, were summarised in tables. The data were also categorised according to age at the cancer treatment time and type of treatment provided. Conclusions: The literature review and LDI patient data revealed that childhood cancer survivors commonly face serious long-term dental issues due to their treatments. These findings highlight the importance of a better understanding of cancer therapy's impact on orofacial development, requiring more attention and support from healthcare professionals, particularly dentists.37 0Item Restricted THE TOBACCO CEMBRANOID (+)-β-CEMBRENEDIOL AND THE PHYTOCANNABINOID (-)-CANNABIDIOL SUPPRESS CANCER PROGRESSION AND RECURRENCE VIA MODULATING TRYPTOPHAN CATABOLISM(University of Louisiana Monroe, 2024-05-22) Mudhish, Ethar; Elsayed, KhalidProstate cancer (PC) and lung cancer (LC) are among the top leading causes of death in United States. Despite completing a therapeutic treatment regimen, including chemotherapies, targeted therapies, surgical excision and/or radiation, patients with PC and LC have a high incidence of recurrence. Recurred tumors usually have a high resistant and aggressive profile and usually lead to high mortality. Unfortunately, there are currently no formally FDA-approved drugs that can prevent cancers recurrence. Therefore, it is crucial to discover and validate safe and effective recurrence suppressors for PC and LC survivors. Studies have shown that the enzyme indoleamine-2,3-dioxygenase (IDO1), usually driving the cellular tryptophan catabolism, plays a critical role in the progression, survival, and motility of PC and LC. IDO1 catabolizes (-)-tryptophan to (-)-kynurenine (Kyn) through the catalytic oxidative cleavage of the indole ring ∆2,3 system. Kyn binding to the aryl hydrocarbon receptor (AhR) lead to the activation lead of several mitogenic downstream pathways, which promote cancer progression and survival. Recently, numerous publications have shed lights on the contribution of tryptophan catabolism in cancer and defined the active and important role of the IDO1-Kyn-AhR axis in promoting oncogenesis. Thus, IDO1-Kyn-AhR validated as a viable molecular target for cancer control. The tobacco cembranoids (+)-(1S,2E,4R,6R,7E,11E)-2,7,11-cembratriene-4,6-diol (β-CBT) and its C-4 epimer α-CBT are critical flavor components that exist naturally in fresh unfermented tobacco leaves. During the commercial tobacco fermentation, a significant portion of tobacco cembranoids, >60%, is intentionally degraded to provide fermented tobacco flavor terpene ingredients with smaller molecular weights. Previous investigations have demonstrated that β-CBT inhibited the invasion of the androgen-independent metastatic PC cell line PC-3M cells and induced tighter intercellular barriers. Further comprehensive studies indicating that β-CBT has in vitro anti-migratory and anti-clonogenicity effects against various human PC cell lines. Additionally, in vivo β-CBT daily treatments significantly suppressed the PC-3M cell locoregional and distant tumor recurrences after primary tumor surgical excision in male nude mice. β-CBT treatments also suppressed the levels of IDO1, TDO2, and Kyn expression levels in PC-3M cells, as well as the systemic levels of the PC biochemical recurrence marker PSA and Kyn in treated animals. These results validated the β-CBT tryptophan catabolism as the main molecular target and highlighted its potential as a novel PC recurrence suppressive lead with high safety and efficacy profiles. IDO1 and AhR are sporadically dysregulated in some PC phenotypes, including metastatic castration-resistance prostate cancer mCRPC. (-)-Cannabidiol (CBD) is a non-psychoactive phenolic secondary metabolite occurring in the leaves and flowers of Cannabis sativa and is assumed a prominent constituent in medical marijuana. CBD formulations approved by the US Food and Drug Administration (FDA) in 2018 and 2019 for treatments of rare epilepsies and tuberous sclerosis complex. These approvals uniquely made CBD exceeds the nutraceutical level and proved its candidacy as a novel drug entity. In vitro studies have demonstrated that CBD exhibited anticancer activities against a variety of malignancies, including human neuroblastoma, medulloblastoma, and lymphocytic leukemia cells, as well as breast, colon, lung, pancreatic, bladder, cervical, endometrial, and prostate cancers. The potential of CBD in cancer therapy has generated considerable interest in recent years, and further research in this area is warranted to establish its clinical efficacy and safety as anticancer drug. This study aimed to comprehensively explore the potential of CBD as a suppressive entity for the progression and recurrence of PC. In vitro experiments revealed that CBD exerted a potent dose-dependent reduction in the viability and colony formation ability of several PC cell lines. Exploring the expression levels of IDO1 and AhR in PC cell lines indicated their dysregulation in metastatic castration-resistant PC (mCRPC) cells. CBD notably suppressed the IDO1 and AhR expression in mCRPC cells. In vivo experiments involving male athymic nude mice xenografted with the mCRPC CWR-R1ca-Luc cells showed that a daily oral dose of CBD effectively suppressed the progression, the locoregional and distant recurrences. Moreover, CBD-treated mice showed a significant reduction in serum Kyn levels, the final product of the IDO1-catalyzed tryptophan catabolism. Knockdown of IDO1 in the mCRPC CWR-R1ca cells resulted in a significant loss of colony formation ability compared to mock-type cells, highlighting the crucial role of IDO1 in mCRPC motility. The catabolism of tryptophan plays a significant role in the pathogenesis of non-small cell lung cancer (NSCLC), a leading cause of cancer-related deaths worldwide. CBD has been found to exhibit anti-proliferative effects on NSCLC cell lines and significantly altered their colony formation abilities. A panel of NSCLC cells has been shown to exhibit dysregulated levels of IDO1 and AhR, both of which are implicated in survival and progression. CBD has been shown to reduce the levels of IDO1 and AhR significantly, thereby providing a promising avenue for curtailing the growth of NSCLC. In light of the presented results, it can be concluded that the modulation of tryptophan catabolism including the IDO1-Kyn-AhR axis is a viable molecular target to control malignancies. The fresh tobacco cembranoid β-CBT presents itself as a promising nutraceutical entity appropriate for use by PC patients to suppress-prevent their disease relapse. Additionally, this study provides support for the notion that CBD serves as a viable natural product lead for regulating the IDO1-Kyn-AhR axis in the context of controlling the progression and recurrence of both PC and NSCLC. β-CBT and CBD are novel anticancer natural products with high translational potential for novel use as prospective cancer recurrence preventing entities.5 0Item Restricted Contribution of Chronic Myeloid Leukaemia Niche to Metastasis and Treatment Resistance(University of Glasgow, 2024) Albilasi, Hakem; Machesky, Laura; Kirschner, Kristina; Jorgensen, HeatherChronic myeloid leukaemia (CML) is a haematopoietic stem cell disorder hallmarked by the Philadelphia chromosome, leading to the formation of the BCR::ABL1 fusion protein and subsequent uncontrolled cell proliferation. Although the advent of tyrosine kinase inhibitors (TKIs) such as Imatinib (IM) has considerably improved patient outcomes, drug resistance and relapse remain a significant challenge. Leukaemia-related deaths and mortalities are often linked to these challenges which are a major cause of human financial and social costs of the disease. The bone marrow microenvironment (BMM) plays a crucial role in normal haematopoiesis and is also the main protection of leukaemic stem cells (LSCs). In CML, the BMM enhances leukemogenesis through an interaction with LSCs, and in turn, LSCs modify the BMM based on their requirements. Mesenchymal stem cells (MSCs) within the BMM are particularly significant, as they can support haematopoietic cells distinctly. The interactions between CML cells and the BMM, including MSCs and the extracellular matrix (ECM), have been shown to influence CML cell proliferation and responsiveness to IM. Moreover, cytoskeletal dynamics also play a crucial role in CML progression and drug resistance. In CML, alterations in cytoskeletal dynamics have been linked to drug resistance and disease progression. Therefore, this thesis will focus on the contribution BMM to these issues, with a particular interest in the role of MSCs and ECM on CML cells. In this thesis, direct contact with MSCs, treatment with MSC-conditioned media, and ECM component exposure were employed to investigate the role of the BMM and MSCs in the proliferation of CML cells and their interaction with the ECM and cell adhesion molecules. Furthermore, the effect of inhibiting specific cytoskeletal components on CML cell behaviour will be examined. The results of this thesis shed light on the complex interplay between CML cells and the BMM, highlighting the critical role of MSCs in modulating CML cell behaviour. While our results did not find a definitive impact of the ECM components on CML cell proliferation or IM sensitivity, the observations suggest that MSCs have the potential to influence the behaviour of CML cells but not definitively affecting their sensitivity to the TKI, IM. Furthermore, inhibiting cytoskeletal components such as the Arp2/3 complex and FAK did not significantly alter the CML cells' contact with MSCs. However, further investigation is necessary to investigate the molecular mechanisms regulating the interaction between CML cells and their microenvironment. Finally, the relationship between CML cells and MSCs, as shown by our transcriptomic analysis, reveals not just alterations in gene expression but also the promise of identifying novel therapeutic targets. The dysregulated pathways in co-cultured K562 cells and MSCs highlight the potential for therapeutic intervention strategies that could disrupt the supportive role of the BMM in CML persistence. The modulation of integrin alpha 9 (ITGA9) expression in K562 cells under co-culture conditions highlights the complexity of CML-MSC interaction and suggests an adaptive mechanism that may contribute to the survival and drug resistance of CML cells within the BMM. Therefore, targeting integrins could potentially enhance the efficacy of existing CML treatments, which in turn, might lead to more effective management of the disease and an increased rate of successful patient outcomes.12 0Item Restricted Understanding the roles of the Reprimo family of proteins in regulating cell behaviour in normal and cancer cells(University of Leeds, 2024-04-23) Arab, Mohammed Mahmoudsami A; Blair, GeorgeThe human Reprimo gene family comprises two genes, RPRM and RPRML. Lack of expression of the RPRM gene has been detected in many human cancer cell lines and tissues. In this study, an immunochemical analysis of the human RPRM was performed. This showed that RPRM could be detected as a FLAG-tagged protein in transfected 293T cells by Western blotting using either an anti-FLAG antibody or (at greatly reduced efficiency) a polyclonal antibody against RPRM. Subcellular fractionation experiments indicated that transiently-expressed RPRM is mainly present in the cytoplasmic fraction of transfected cells, however fractionation of the cytoplasm into cytosol and membranes revealed a predominant association of the 18kDa RPRM form with intracellular membranes. Analysis of several human cell lines for the endogenous RPRM forms also revealed a membrane location of the 18kDa RPRM form. Localisation of RPRM by immunocytochemistry also revealed it to be associated with membranous structures, that co-localised with the Golgi/endoplasmic reticulum. Immunohistochemical studies on normal and cancerous human tissues (including breast, pancreas and colon) provided further evidence for a membrane location of RPRM. Studies were performed on the reduction of the endogenous RPRM forms by RNA interference and the impact of RPRM reduction on the cell cycle. These studies revealed small, although significant effects of RPRM on the G1 to S and the of G2 to M phase transition. Over-expression of RPRM by transient transfection of MCF7 breast cancer cells or 293T kidney cells resulted in a block to the G2 to M phase. In summary, RPRM exists in a number of forms related by differential posttranslational modification, probably principally N-glycosylation. It has a major location with intracellular membranes and influences the cell cycle in different ways, dependent on the level of RPRM expression. However the link between RPRM membrane association and the cell cycle remains to be elucidated.45 0