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Item Embargo The Impact of Child Protection Training on Nurses’ Self-Efficacy in Detection and Reporting of Child Abuse in Saudi Arabia(Trinity College Dublin, the University of Dublin, 2024) Almutairi, Albandari; Corry, Margarita; Hughes, MaryBackground: The World Health Organisation (WHO) has reported that child abuse and neglect constitute major international health problems that cause unacceptable morbidity and mortality. These forms of maltreatment can have severe physical and psychological effects on children’s development that persist into adulthood. Early detection of abuse and removing children from potentially hazardous and perilous conditions pose significant challenges. Nurses who care for children and families are uniquely positioned to identify children at risk of abuse and neglect during visits and have the opportunity to initiate interventions to prevent future harm. However, barriers such as a lack of experience, training, and confidence in handling abuse cases exist. Several training programmes have been developed to enhance nurses’ abilities to recognise and report child abuse. Studies demonstrate that these training programmes effectively improve nurses’ awareness, detection, and reporting of child abuse and neglect. In Saudi Arabia, The National Family Safety Program provides non-mandatory training to healthcare practitioners to support them in identifying and reporting child abuse cases through the Child Protection Multidisciplinary Training Programme (CPMTP) in multiple healthcare sites. The concept of self-efficacy has been used to assess the outcomes of clinical training programmes in other fields, as positive effects on self-efficacy should translate to desirable practice patterns. To date, few studies have explored the impact of child abuse training on nurses’ self-efficacy in recognising and responding to known and suspected cases of child abuse and neglect. Furthermore, none of these studies have been conducted in Saudi Arabia. Aim: To explore the impact of child abuse training on nurses’ self-efficacy in recognising and responding to known and suspected cases of child abuse and neglect (CAN) in Saudi Arabia. Methods: This study was conducted across three regions of Saudi Arabia, with participation from nurses caring for children, using the Child Abuse Neglect Reporting Self-Efficacy Questionnaire (CANRSEQ). The questionnaire was administered in online and hard-copy formats across all three regions, with a completion time averaging under 20 minutes. Data were analysed using descriptive statistics and correlation analyses using Statistical Package for Social Sciences (SPSS) version 28 software. Findings: The response rate was 77% (n = 247) among staff nurses. The majority (75.5%) had not reported any cases of child abuse or neglect in the past. The findings indicate that 73.2% (n = 164) did not receive any formal CAN cases. Additionally, 19.2% (n = 43) of the respondents had undergone formal training sessions concerning CAN, and 10.4% (n = 23) attended the CPMTP provided by NFSP. The study’s findings showed a significant difference in self-efficacy expectations (EEs) on the SE-CAN scale. However, there was no significant difference in outcome expectation (OE) scores between those who attended CAN training and those who did not, as measured by the OE-CAN scale. The respondents generally reported being more confident in handling known abuse cases than in suspected abuse cases. Self-efficacy in detecting and reporting CAN differed among education groups when measuring the relationship between personal characteristics and professional self-efficacy in nurses. Those who had a postgraduate degree and attended a training programme reported a statistically significant higher self-efficacy score (i.e., EES, EEK, OE; mean = 3.4864 ± 0.56619) compared to those with a bachelor's degree (mean 3.2337 ± 0.59468, p < .05). Conversely, no significant differences were found in self-efficacy based on other personal characteristics such as age, gender, or marital status (p > 0.05). Regarding knowledge of the law, most of the participants, 70.7% (n = 164), believed they had a legal obligation to report cases where a child or young person was at risk of physical or sexual abuse. However, the lowest percentage, 42.1% (n = 98), was recorded for cases where a prenatal report was made, but the birth mother did not receive adequate support services. Conclusion: This study is the first in Saudi Arabia to investigate the impact of child abuse training on nurses’ ability to identify and respond to CAN. The results provide an important contribution to our understanding of nurses’ self-efficacy in managing such cases, as they highlight the specific areas where nurses feel most confident and capable. Additionally, the findings shed light on potential areas for improvement and the need for targeted interventions to enhance nurses’ self-efficacy in specific aspects of CAN. The underreporting of child abuse is attributed to various barriers, but training programmes can significantly improve nurses’ confidence in reporting abuse cases. Notably, nurses are more confident in reporting known cases rather than suspected cases, and the ability to detect abuse is dependent on the type of abuse being investigated (physical, emotional, and sexual abuse). Therefore, training programmes must enhance nurses’ confidence and provide them with clear guidelines for mandatory reporting and support in navigating these challenges.8 0Item Restricted Electron FLASH beams from a modified Elekta Precise LINAC: characterisation and dosimetry(University College Dublin, 2024-12) Mousli, Majed Hussain; Vintró, Luis LeónRadiotherapy treatment aims to administer a substantial dose of ionising radiation to the tumour volume while limiting exposure to surrounding healthy tissue to reduce potential side effects. Typically, daily fractionated doses of about 2 Gy each, extended over an average of around 20 fractions (typically ranging from 5 to 39 fractions) across diverse tumour sites are employed. This conventional dose fractionation utilises techniques like 3D conformal radiotherapy (3DCRT), intensity-modulated radiotherapy (IMRT), volumetric modulated arc therapy (VMAT), and stereotactic ablative radiotherapy (SABR) to deliver the dose, with average dose rates varying from 6 Gy/min (using conventional flattened beams) to 24 Gy/min (employing flattening filter-free (FFF) beams) to expedite treatment duration and enhance patient comfort. Recently, FLASH radiotherapy, which makes use of ultra-high dose rates (UHDR) of >40 Gy/s to irradiate targets, has been the subject of much research. This approach holds the potential to augment the therapeutic ratio and significantly reduce treatment times, where the therapeutic ratio signifies the balance between tumour control probability (TCP) and normal tissue control probability (NTCP) likelihood. The main aim of this thesis was to investigate the possibility of making use of a suitably modified standard radiotherapy linear accelerator (LINAC) to produce FLASH level electron beams, and to characterise the resulting beams with a range of different detectors, including a novel inorganic scintillator detector. As a first step in this study, a Monte Carlo (MC) model of the Elekta Precise LINAC was developed using the EGSnrc MC code to characterise conventional systems utilising 10, 12, and 15 MeV electrons. The MC model was employed to investigate the effect of making changes to the LINAC towards achieving FLASH-level beams, and to assess detector responses, encompassing dose, dose linearity, and dose rate dependence when subjected to FLASH beam conditions. The model's validation was performed against an advanced Markus ionising chamber (AMIC) and Gafchromic EBT XD film measurements for both conventional and modified LINAC set ups. The results showed very good agreement between simulated and measured data, with depth dose percentages (PDD), as well as the relative distances R90, R50, and Rp giving variations within ±1 mm for both conventional LINAC across the 10, 12, and 15 MeV electron energies and modified LINAC for the 12 MeV electron energy. Bremsstrahlung contamination was found to be 1% and 2% for conventional LINAC and modified LINAC, respectively. The beam profiles showed deviations of ±2% (cross-plane) and (in-plane) for the 12 MeV electron beam under conventional and modified LINAC conditions. In conclusion, the MC model demonstrates robust agreement between measured and calculated data for both conventional and modified LINAC settings, offering potential utility in validating diverse dosimetry detectors for assessing dose, dose rate, and dose per pulse (DPP) dependencies under FLASH irradiation. The second objective was to characterise multiple dosimetric detectors using a modified Elekta Precise linear accelerator (LINAC) to generate an electron FLASH beam for radiotherapy (eFLASH-RT), achieving radiobiological research-level FLASH conditions, and to compare the results with those predicted using Monte Carlo (MC) simulation. The 12 MeV electron beam was adapted into an eFLASH setup by adjusting beam parameters and positioning the carousel at an open port with primary and secondary scattering foils. A microcontroller unit (MCU) circuit was used to control the pulse count. Gafchromic EBT-XD films were used to established PDD, beam profiles and DPP, and these were validated against MC simulations. Both conventional beam radiotherapy (CONV-RT) and eFLASH-RT showed that the simulated and measured data for reference depths R90, R50, and Rp agreed to within ± 1 mm. Gamma analysis, with passing rates >95% (gamma criteria 3%/3mm), showed good PDD and beam profile agreement. DPP changed with SSD (54 to 150 cm) from 0.2 to 0.025 cGy/pulse in CONV-RT to 60 to 8 cGy/pulse in FLASH-RT, with negligible differences (< 2 cGy) between MC and each detector. By taking secondary scattering foils out of the beam path and reducing the SSD, the model predicted that dose rates in excess of 40 Gy/s could be achieved. This was confirmed by using the PTW AMIC detector and EBT XD films. The model was used to analyse parameters like PDD, large-field beam profiles, dose, dose rate, and DPP dependencies. The results of this study demonstrate that the modified LINAC can be used as a tool to assess dosimetry detectors for FLASH radiotherapy, potentially aiding online real-time radiation dose evaluation during therapy and facilitating radiobiological investigations into eFLASH-RT's cellular impact. The final part of the thesis investigated the use of a novel inorganic scintillating detector (ISD) employing Gd2O2S:Tb with a short temporal resolution (2.8 ms), as well as a plastic scintillation material (PSD), for real-time dosimetry in ultra-high dose rate applications. The modified LINAC was used to deliver a 12-MeV electron FLASH beam (eFLASH) at an average dose rate of 85 Gy/s. The response of the scintillation dosimeters (operated using the HYPERSCINT radiation platform) were compared to that of the AMIC detector across various field sizes and electron applicators to assess linearity, repeatability, DPP, and output factors at a 4-cm water-equivalent depth. The plastic (PSD) and inorganic scintillator detector (ISD) measurements were validated against EBT-XD Gafchromic films and Monte Carlo (MC) simulations. Relative IC measurements exhibited linear behaviour, with PSD and ISD responses differing within 1% and 14%, respectively, over a 50 to 7000 cGy range. PSD maintained linearity with increasing pulse repetition frequency (PRF) up to 172 Hz (max value 1.1), while ISD response at 40, 72, and 172 Hz yielded linearity indices of 1.2, 1.35, and 1.24. PSD and ISD showed consistent dose responses under ultrahigh dose rate conditions, agreeing within 4% with EBT-XD film readings. Furthermore, DPP linearity at different SSDs was studied, yielding favourable agreement for 5 cm2 field sizes using EBT-XD film, AMIC, and MC models. The novel PSD and ISD detectors, integrated into the HYPERSCINT platform, demonstrated a robust response to 12 MeV eFLASH beam irradiation, accommodating DPP of up to 0.65 Gy and an ultra-high dose rate of 85 Gy/s. This work will establish a versatile dosimetry platform that will be applicable to various research domains, including in vitro and in vivo radiobiology investigations of ultrahigh dose rate irradiation.10 0Item Restricted The Role of e-leadership on Remote Decision Making: Key Learnings from the COVID-19 Pandemic(Cork University Business School, 2024) Alsqeah, Latifah; Adam, Frederic; Treacy, StephenThis thesis investigates the role of e-leadership competencies in decision-making processes within private businesses during the COVID-19 pandemic. The pandemic forced a rapid transition to remote work, necessitating a deeper understanding of how leaders adjust to virtual environments and employ e-leadership practices. Despite the growing relevance of remote work, empirical studies on e-leadership remain scarce, particularly in the context of decision-making—a core leadership responsibility. This research addresses this gap by exploring the importance and impact of e-leadership competencies on remote decision-making and the value propositions these competencies present. The primary objective of this study was to investigate how e-leadership competencies influenced decision-making among private-sector leaders in Saudi Arabia during the COVID-19 crisis. A qualitative field study was conducted through interviews with 19 leaders from various private sectors, including software development, logistics, oil mining, and training services. These interviews provided in-depth insights into how leaders faced the challenges of remote decision-making during the pandemic. The findings indicated that leaders recognised two key e-leadership competencies, e-communication and e-technology, as crucial for facilitating remote decision-making. These skills enabled managers to maintain clear and consistent communication with their staff, utilising advanced technology to manage remote work successfully. Critical competencies such as e-trust, e-team, and e-change were also recognised as essential, highlighting their significance in building trust, managing teams, and supporting change in a remote environment. Interestingly, 70% of participants considered e-social competency crucial, yet not all leaders agreed, indicating differing viewpoints on the role of social skills in e-leadership. Through this analysis, two theoretical models emerged from the findings: i.A preliminary model of e-leadership competencies’ impact on remote decision-making processes. ii.A preliminary model of the value propositions of remote decision-making. These models identified seven key impacts influencing decision-making in a remote work environment, alongside five primary value propositions related to remote decision-making. This research makes several significant contributions to the IS field and practice. Firstly, it empirically validates and extends Roman et al.'s (2019) e-competency framework by applying it to the context of remote decision-making during the COVID-19 pandemic. This application resulted in the development of two preliminary models that explain the impact of e-leadership competencies on decision-making processes in remote work environments. Secondly, the study fills a significant gap in the literature by identifying new value propositions associated with remote decision-making. Lastly, the research broadens the understanding of e-leadership in the context of private businesses, offering practical implications for managing remote work during disruptions and contributing valuable insights to the literature on business continuity and e-leadership. This thesis provides a comprehensive analysis of how e-leadership competencies influence remote decision-making in a disrupted remote work environment. The findings highlight the essential role of e-competencies in navigating the challenges of remote work, offering both theoretical advancements and practical guidance for leaders facing similar situations in the future. As organisations continue to adapt to the evolving landscape of remote working, the insights from this study will prove valuable in understanding and enhancing the effectiveness of remote decision-making.16 0Item Restricted Nurses’ Experiences of Breaking Bad News in Oncology Settings With Adolescents and Young People(Trinity College Dublin, 2024-08-22) Alyami, Malak; Murphy, MaryanneABSTRACT Background: Oncology nurses communicate with adolescents and young adults (AYAs) at the end of life to break bad news, communicate important information, or gain insight into the patient’s condition, anxiety, fear, or pain. However, oncology nurses face many barriers to communicating with AYAs to break bad news at the end of life, so identifying these barriers is an essential step in overcoming them. What barriers do oncology nurses face when communicating with AYAs to break bad news at the end of life? Aim: To conduct a systematic review examining barriers oncology nurses face when communicating with AYAs to break bad news at the end of life. Methods: A systematic review question was developed, a search strategy was defined, and inclusion and exclusion criteria were applied to the search results. Afterwards, a quality assessment of the included studies was conducted, and data extraction was conducted according to JBI standards and protocol. The meta-aggregative approach was applied, and the extracted data were grouped into prominent themes. Results: The search and selection strategy resulted in 2,080 studies to which the inclusion and exclusion criteria were applied. After filtering the studies, 20 remained. Four met the high-quality assessment criteria and were included in the thematic analysis and meta-aggregative process. The analysis and synthesis process resulted in the emergence of five main themes related to the barriers faced by oncology nurses in breaking bad news to AYAs at the end of life: 1) communication skills and experiences of oncology nurses, 2) individual barriers, 3) professional barriers, 4) institutional barriers, and 5) societal barriers to breaking bad news. Conclusions: Oncology nurses face many barriers related to their lack of competence and effective communication skills. These barriers may be professional or related to the lack of protocols for breaking bad news and communicating with AYA oncology patients. They may also be due to institutional barriers resulting from inadequate resources, time, and nurses. In addition, the relationships between nurses and multidisciplinary teams, the overlap of tasks, and the lack of clarity of roles in the process of breaking bad news constitute barriers to the success of the communication process, in addition to the linguistic and cultural diversity between nurses, patients, and their families, which constitute major challenges in the communication process.12 0Item Restricted Pedagogy Potential of Social Media in Saudi Arabia Curricula: students' and teachers' perceptions and practices(University of Limerick, 2024-05) Alhassan, Yousef; Murray, Liam; Giralt, MartaSocial media tools are commonly used in everyday life in the 21st century. Social media has influenced many areas in our lives including education. Social media is widely researched in the area of English as a foreign language (EFL). Research into social media as a supportive tool in the curriculum has been reported with different perspectives. Some scholars (Mohammed et al., 2023; Lashari et al., 2023) have found it useful in improving the performance of English language learners and others have indicated (Nkhi, 2023; Asafo-Adjei, 2023) negative impacts of integrating social media in EFL classrooms. The present doctoral research aimed at exploring the perceptions and practices of teachers and students at 3rd level education towards using social media as a tool in curricula in Saudi Arabia. This research targeted learners and teachers of EFL in Saudi Arabia. The study investigation included the student use of social media, the student’s perceptions of social media, teacher’s use of social media and the teacher’s perceptions surrounding social media. Moreover, this research suggested several practices that can be useful for teachers and learners of EFL. A mixed method approach of quantitative and qualitative approaches was adopted to collect the data for the study using questionnaires and semi-structured interviews for the two targeted groups, teachers and students. These tools are used to explore the perceptions and attitudes of the participants towards using social media as a tool in EFL classrooms. The data were analysed using statistical analysis for the quantitative data and thematic analysis for the qualitative data. Overall, the results of the research indicated that using social media in curricula appeal to both teachers and students with concerns regarding distraction that social media may cause and the possibility to find appropriate approaches that can be adopted to implement social media in EFL classrooms.11 0Item Restricted Myeloid Differentiation of Resident Vascular Stem Cells (mVSCs) into Macrophage-Like Cells (MLCs) in Response to Ox-LDL and Cholesterol(dublin city University, 2024-09) Muhayla Alanazi; Cahill, paulA hallmark of subclinical atherosclerosis is the build-up of vascular smooth muscle cell (SMC)- like cells and non-SMC cells, particularly macrophage-like cells (MLCs), which cause intimal medial thickening (IMT), lipid buildup, plaque development and blood flow restriction that can lead to a heart attack or stroke. Previous cell fate mapping studies have implicated a putative role for SMCs and resident S100β vascular stem cells (mVSCs) in promoting lesion formation, prior to plaque development. The specific aim of this study was to assess whether murine and human S100β vascular stem cells are capable of myeloid differentiation to MLCs when exposed to atherogenic stimuli cholesterol, oxidized LDL (ox-LDL) in vitro before their response was compared to murine and human SMCs. MLC phenotype was determined by morphological changes, immunocytochemical and immunophenotyping of macrophage surface markers using flow cytometry, in addition to histochemical analysis of lipid droplets and real-time PCR analysis of macrophage phenotypic markers. The data reveal that murine and human SMC-like cells exposed to atherogenic stimuli in vitro increased their expression of myeloid phenotypic markers, typical of MLCs. Moreover, S100β vascular stem cells were also capable of myeloid differentiation but to a greater extent than SMCs in culture in response to the same atherogenic stimuli in vitro. These data suggest that S100β vascular stem cells may represent a novel target for therapeutic intervention to reduce the number of MLCs in atherosclerotic lesions.3 0Item Restricted Systematic Review The Impact of Sedentary Behaviour on Health Outcomes in Chronic Obstructive Pulmonary(Galway University, 2024-08-25) Alshareef, Ibrahim; Said, Marwa; Marah, FeghaliThe Impact of Sedentary Behaviour on Health Outcomes in Chronic Obstructive Pulmonary3 0Item Restricted Application of a Targeted Proteomics Strategy for the Development of a Blood Protein Signature for Breast Cancer Stratification and Prognostication(University College Dublin, 2024-05) Metwali, Essraa; Pennington, StephenBreast cancer (BC) is a common malignancy in women worldwide, affecting roughly 3 million and of these 15 to 20% of cases are triple-negative breast cancer (TNBC). In comparison to other types of breast cancer, TNBC is distinguished by the absence of estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2. This makes it a particularly challenging therapeutic target due to its lack of specific receptor signalling pathways that can be targeted therapeutically. Patients with TNBC often experience systemic relapse and poor prognosis. The traditional treatment guidelines for early TNBC are based on chemotherapy, surgery, and radiation to prevent disease recurrence. Although novel treatments are constantly being introduced for BC, conventional cytotoxic chemotherapy is the primary treatment option for TNBC. Therefore, the approach to treating early TNBC has shifted towards neoadjuvant treatment (NAC), given to the patient before surgery. This approach aims to reduce tumour size, thus improving the chances of surgical intervention and reducing the risk of recurrence. Research has proven that NAC prior to surgery is associated with 30% of patients achieving pathological complete response (pCR), directly linked to improved survival rates. Thus, a novel predictive biomarker of NAC response is paramount to selecting patients with good prognoses, de-escalating systemic treatment to minimize chemotherapy toxicities, and increasing the likelihood of achieving pCR. So, the objective of this study is to apply mass spectrometry-based proteomics to serum samples from TNBC patients (both those who achieved pCR and those who did not) who have received NAC to identify i) biomarkers that might measure response to NAC, ii) biomarkers that might correlate with the extent of residual disease. Serum samples from TNBC patients were provided by the Royal College of Surgeons in Ireland (RCSI), following ethical guidelines and approval. Patient samples included in this study had undergone NAC treatment, anthracyclines and taxanes, for approximately 3-5 months before surgery. Patients were then distinguished based on whether they achieved pCR during surgery or not. In this study, there are two essential proteomic approaches to identify and initially verify biomarkers: discovery and targeted proteomics. Label-free LC-MS/MS based protein discovery was undertaken on depleted serum samples from both patient groups, revealing a total of 19 significant proteins and 124 unique peptides, that could be beneficial for classifying TNBC patients who would achieve residual disease after the NAC. These candidate biomarkers along with those identified in literature reviews, and tissue-based gene expression data (Human Protein Atlas), are being incorporated into the development of targeted proteomics multiple reaction monitoring (MRM) assays. Robust MRM assays were successfully designed and developed for a total of 73 proteins represented by x 84 peptides to facilitate further verification and evaluation of these proteins of interest. MRM measurements of the 73 candidate biomarker proteins were performed on serum samples acquired from the same patients used in the discovery approach, verifying the significant changes in expression between patients with and without disease recurrence. The results identified a subpanel of 6 proteins, with 5 identified in the discovery approach and one from the literature, indicating potential for prognosis assessment. The second part of the study utilized three different TNBC cell lines to assess the robustness of the developed MRM assays. Several detectable proteins were identified across the three cell lines, CAL-5, MDA-MB231 and MDA-MB 436 cells. Moreover, the successful performance of the MRM assays led to the identification of three proteins that were significantly expressed across the three cell lysates, offering insight into potential functions and aiding in understanding the variation observed in TNBC. All protein measurements reported in this study were achieved with a high degree of reproducibility resulting from the technical rigor under which all phases of the experimental process were undertaken. Overall, these studies highlight the utility of mass spectrometry-based proteomics to address key unmet clinical needs in TNBC. Namely, the need for novel serum-based protein biomarkers to distinguish and early prognostication between TNBC patient groups who are being treated with NAC. Besides, the development of a robust MRM method for identifying protein biomarkers associated with NAC responses in both TNBC group may serve as a valuable resource for ongoing research and in the BC research community internationally.4 0Item Restricted Exploring Challenges and Issues Faced by Faculty Members in Utilizing Blackboard within Higher Education in the Kingdom of Saudi Arabia (An Empirical Study of King Saud University)(LIMERICK UNIVERSITY, 2024) Alqahtani, Mohammed; Murray, LiamThis study addresses a crucial issue pertaining to the influence of the design and user interface of a learning management systems (LMSs), known as Blackboard, on the pedagogical performance of faculty members at King Saud University in terms of their employed teaching methods. The study considers system design and user interface as independent variables, while teaching methods are regarded as the dependent variable. The research aims to provide answers to the following specific inquiries: - How do faculty members at Saudi universities perceive Blackboard as a digital platform that replaces traditional teaching methods? - How do faculty members at Saudi universities critically evaluate the design of the Blackboard system? - How do faculty members at Saudi universities critically evaluate the user interface of the Blackboard system? Due to the intricate nature of human-computer interactions, interview alone may not offer sufficient information. Consequently, the researcher adopted mixed methodology research techniques, which involved conducting interviews with five teachers from the targeted institution and gathering 77 teacher responses through online questionnaires. The study received approximately 212 responses to the three open-ended questions on the survey which was distributed with the questionnaire. The data collected from both interviews and surveys facilitated a comprehensive analysis of the strengths and weaknesses of the system design and user interface of each LMS in relation to the employed teaching methods. The findings of the study underscore the substantial impact of LMS designs on system effectiveness and provide evidence of how variations in system design influence the overall user experience. The conclusions assert that the Blackboard system, particularly at the University of King Saud, significantly contributes to the educational process by enhancing both learning and teaching. The researcher conducted interviews with five teachers who utilized the Blackboard system during the coronavirus pandemic. The data were tabulated using NVivo software, which is regarded as being one of the most effective programs for analyzing text-based data as it has the ability to enhance the qualitative research process by expediting the analysis of textual data, which would otherwise be done manually. In conclusion, the recommendations include prioritizing multilingual support, implementing thorough training for instructors, ensuring prompt system updates and technical support, expanding language alternatives, and establishing a user feedback mechanism within Blackboard. Moreover, there is a call for a longitudinal study on LMS roles beyond the pandemic. This emphasizes the importance of researching how adaptable LMS is to evolve educational requirements and assessing its support for diverse learning methods over time.3 0Item Restricted Investigating the contributions to cell function of the different Swi-Snf complex subunits in Saccharomyces cerevisiae(Trinity college Dublin, 2024) Aljaeed, Hadel Mosfer; Fleming, AlastairThe eukaryotic genome is packaged as a DNA-protein structure known as chromatin. The basic subunit of chromatin is the nucleosome, which contains two copies of each of the core histone proteins H2A, H2B, H3, and H4, around which is wrapped 146 - 147bp of DNA. This structure is generally repressive for processes such as gene transcription that require access to the DNA. However, chromatin is dynamic. ATP dependent chromatin remodelling complexes can act to open or close this structure to enable or deny access to the DNA as and when required. The first ATP-dependent chromatin remodelling complex discovered was the Swi-Snf complex found in the yeast, Saccharomyces cerevisiae. This multi-subunit complex is best characterized as an activator of transcription. However, the precise role of each of the Swi-Snf subunits in transcription is not well understood. The first goal of the study was to investigate the role of each Swi-Snf complex subunit in cell function by examining the phenotypes in various Swi-Snf subunit mutants. Specifically, I examined snf2, swi3, snf5, snf6, snf11, snf12, taf14, and swp82 deletion mutants in addition to a snf2K798A catalytically dead mutant. Unfortunately, I was unable to obtain a SWI1 mutant to include in my analysis. My repeated gene deletion attempts failed which I hypothesised was due to the swi1 deletion mutant being either too sick to recover or being inviable. Also, my attempt to create a Swi1p anchor-away strain was not successful. Considering there are reports of swi1 mutants in the literature, I would suggest that the ability to obtain a viable swi1 mutant is strain specific and was incompatible with the strain backgrounds I used. The phenotypic analysis revealed that some Swi-Snf subunit mutations significantly affected doubling time and growth, while others had a minimal impact. For example, the greatest growth defect was found in snf2 and snf5 mutant strains. The snf2 and snf2K798A mutants were not always similar and displayed many distinct phenotypes. III When cell morphology was examined, the data revealed that the snf6 mutant displayed an elongated cell morphology, while snf2, snf2K798A, and snf5 strains had a clumpy cell morphology. According to spot test results using different reagents and different temperatures in liquid and solid media, the mutants again showed differences in their response to DNA-damaging reagents and cell wall stress. Together, the phenotypic tests indicated that the different subunits can possess diverse and distinct activities in multiple cellular processes, such as controlling cell size and proliferation, as well as influencing the cellular response to DNA damage. RNA-Seq analysis was used to compare the transcriptomes of each mutant strain to wt and to each other. Since the Swi-Snf complex functions as a co-activator of gene transcription, I expected most genes in each mutant to be downregulated. Surprisingly, this was not the case. For example, snf2 mutants, which should completely cripple the remodelling activity of the complex, had a similar number of genes up-regulated as were downregulated. This suggests SNF2 plays an equal role in the negative regulation of transcription as it does in positively regulating gene transcription. Furthermore, the swi3 deletion mutant showed almost twice as many genes were upregulated suggesting the major role of SWI3 is in the repression of transcription. Thus, the data suggests that the Swi-Snf co-activator complex can be considered to equally be a co repressor of transcription. The transcriptome data also revealed that even among Swi-Snf subunits within the same module, there were large variations in the number of genes regulated by each subunit, as well as differences in how these functionally related subunits contribute to the transcription of these genes. For example, whereas one subunit might be required for activation of a gene, another subunit could play a role in repression of the same gene. Most strikingly, there was only a minimal overlap (57 genes) in the transcriptomes of snf2, swi3, snf6 and snf5 mutants which, between them, are subunits representative of each of the Swi-Snf submodules. Together, this suggests each subunit can exert widespread distinct regulatory effects upon positive and negative gene IV transcription when functioning either within the Swi-Snf complex or, most intriguingly, when functioning outwith the complex. Analysis of published global sites of occupancy across the genome for the various Swi Snf subunits revealed that each subunit occupied a distinctly different number of sites. Swi3p occupied the greatest number of sites compared to other subunits (980 sites), whereas Taf14p had the most unique occupancy profile in terms of its presence at sites other than protein coding genes. Interestingly, Snf2p was found located most frequently across gene coding regions compared to its occupancy at promoters. This was surprising, as the best characterized role for Swi-Snf has been in remodelling nucleosomes at gene promoters to enable transcription initiation. This latter data might suggest a primary role for Snf2p dependent chromatin remodelling by Swi-Snf is during transcription elongation. Importantly, there was only a small overlap in the number of sites occupied by all the Swi-Snf subunits analysed. This is in support of Swi Snf subunits being able to function independently from each other and outwith the confines of the Swi-Snf complex. Swi-Snf subunit occupancy data was also correlated with the lists of genes up and downregulated in each mutant. The goal was to determine the specific genes that are directly controlled by each Swi-Snf subunit, and to assess whether Swi-Snf subunits could be working independently from each other within or outwith the complex. The data revealed that the correlation between Swi-Snf subunit occupancy and genes shown to be influenced by the particular subunits was poor. This could indicate that the major impact upon transcription is indirect or works over long-range distances. Conversely, the results could be due to limitations of the ChIP technique in fully identifying the sites of protein occupancy, or the sites of occupancy of the subunits are not occupied under the glucose-grown conditions used in this study. Indeed, performing ChIP for the subunits under multiple growth and stress conditions might be needed to identify the full suite of sites occupied by Swi-Snf as its recruitment at certain target sites will be cell signal specific. V The second part of my project examined the interplay between the Swi3p subunit of the Swi-Snf co-activator complex and the Cyc8p subunit of the Tup1-Cyc8 co-repressor complex. This interaction was chosen for analysis because previous unpublished data, which was confirmed in this study, revealed that Cyc8p levels were undetectable by Western blotting in a swi3 deletion mutant. Importantly, since CYC8 transcription levels were unaffected in the swi3 mutant, this suggests that the impact of the SWI3 gene deletion upon Cyc8p levels was occurring at the level of the Cyc8 protein. Furthermore, the SWI3 gene was shown to be responsible for repressing almost twice as many genes as it activates, thus implicating Swi3p as having a large role in mediating gene repression. Firstly, to investigate this potential interplay between SWI3 and CYC8 the swi3 transcriptome was compared to that of the swi3 cyc8 double mutant. This was to test the prediction that the swi3 mutant, in which Cyc8p was potentially absent, would have a transcriptome similar to that in a swi3 cyc8 double mutant. However, the results showed that this was not the case. The data showed that CYC8 could still negatively influence transcription of genes in the swi3 mutant suggesting that despite the abundance of Cyc8p in this mutant being below the detection threshold of western blotting, Cyc8p was still present at a level that could influence gene transcription. Secondly, by comparing the transcriptomes of swi3, cyc8 and swi3 cyc8 mutants, the data revealed that gene repression by SWI3 could function both independently of CYC8, and synergistically with CYC8. These findings therefore showed that the Swi3p subunit significantly contributes to the repression of gene transcription via Cyc8p-dependent and independent mechanisms. Finally, co-immunoprecipitation was used to confirm a direct interaction between Swi3p and Cyc8p. The results also suggested that the Swi3p and Cyc8p interaction might be mediated by Swi3p and Cyc8p whilst these subunits are residing within their respective Swi-Snf and Tup1-Cyc8 complexes. VI Overall, these data suggest wide-ranging unique roles for the individual subunits of the Swi-Snf complex that may be functioning when these proteins are residing within or out with the complex. The data also implicates the Swi-Snf co-activator complex as having an almost equal role in mediating gene repression, and that Swi3p plays the greatest role in this negative regulation of transcription. Finally, I have revealed evidence of a direct physical interaction occurring between the Swi-Snf and Tup1-Cyc8 complexes mediated by the Swi3p and Cyc8p subunits. Together, these data oppose the generally accepted consideration of these complexes independently acting as predominant activators or repressors of transcription. Instead, the data might suggest these complexes should be considered as intimate partners which work together to ensure gene transcription is accurately and appropriately positively and negatively regulated in response to the changing environment.2 0