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This paper focuses on the use of Izak9 in promoting the development of mathematical reasoning and Math-Talk in an Irish classroom. Recent national and international assessments have shown many positive results in relation to the mathematical learning of Irish pupils (Shiel et al., 2014; Perkins & Clerkin, 2020). However, the skill of reasoning is a relative weakness among this cohort (Perkins & Clerkin, 2020). Reasoning is highlighted as one of the four ‘elements’ of the proposed new Primary Mathematics Curriculum (PMC), which is currently under development (National Council for Curriculum and Assessment (NCCA), 2016; NCCA, 2017). This study was qualitative in nature. The research methods utilised were focus group interviews and observations of mathematics lessons. Findings of this study demonstrated that mathematical reasoning skills were promoted in Izak9 lessons, specifically the exploration of patterns and the justification of mathematical processes and solutions. In addition, a Math-Talk environment was also supported through the use of the resource.

In lung cancer radiotherapy (RT) high doses are delivered to the heart. We found significant associations between death rate (DR) and high left atrial wall (LAWall) volumes receiving 63-69Gy (LAWallV63-69) and any new or worsening ischaemic or rhythm ECG changes within 6 months of RT in a prospective isotoxic dose-escalation trial of 78 locally advanced non-small cell lung cancer (LA-NSCLC) patients. Here we aim to validate these findings in an independent retrospective cohort.

The nuclear pore complex (NPC) machinery is emerging as an important determinant in the maintenance of genome integrity and sensitivity to DNA double-strand break (DSB)-inducing agents, such as ionising radiation (IR). In this study, using a high-throughput siRNA screen, we identified the central channel NPC protein Nup54, and concomitantly its molecular partners Nup62 and Nup58, as novel factors implicated in radiosensitivity. Nup54 depletion caused an increase in cell death by mitotic catastrophe after IR, and specifically enhanced both the duration of the G2 arrest and the radiosensitivity of cells that contained replicated DNA at the time of IR exposure. Nup54-depleted cells also exhibited increased formation of chromosome aberrations arisen from replicated DNA. Interestingly, we found that Nup54 is epistatic with the homologous recombination (HR) factor Rad51. Moreover, using specific DNA damage repair reporters, we observed a decreased HR repair activity upon Nup54 knockdown. In agreement with a role in HR repair, we also demonstrated a decreased formation of HR-linked DNA synthesis foci and sister chromatid exchanges after IR in cells depleted of Nup54. Our study reveals a novel role for Nup54 in the response to IR and the maintenance of HR-mediated genome integrity.

Purpose To determine the relative abilities of compartment models to describe time-courses of 18F-fluoromisonidazole (FMISO) uptake in tumor voxels of patients with non-small cell lung cancer (NSCLC) imaged using dynamic positron emission tomography. Also to use fits of the best-performing model to investigate changes in fitted rate-constants with distance from the tumor edge. Methods Reversible and irreversible two- and three-tissue compartment models were fitted to 24,662 individual voxel time activity curves (TACs) obtained from tumors in 9 patients, each imaged twice. Descriptions of the TACs provided by the models were compared using the Akaike and Bayesian information criteria (AIC and BIC). Two different models (two- and three-tissue) were fitted to thirty measured voxel TACs to provide ground-truth TACs for a statistical simulation study. Appropriately scaled noise was added to each of the resulting ground-truth TACs, generating 1000 simulated noisy TACs for each ground-truth TAC. The simulation study was carried out to provide estimates of the accuracy and precision with which parameter values are determined, the estimates being obtained for both assumptions about the ground-truth kinetics. A BIC clustering technique was used to group the fitted rate-constants, taking into consideration the underlying uncertainties on the fitted rate-constants. Voxels were also categorized according to their distance from the tumor edge. Results For uptake time-courses of individual voxels an irreversible two-tissue compartment model was found to be most precise. The simulation study indicated that this model had a one standard deviation precision of 39% for tumor fractional blood volumes and 37% for the FMISO binding rate-constant. Weighted means of fitted FMISO binding rate-constants of voxels in all tumors rose significantly with increasing distance from the tumor edge, whereas fitted fractional blood volumes fell significantly. When grouped using the BIC clustering, many centrally-located voxels had high fitted FMISO binding rate-constants and low rate-constants for tracer flow between the vasculature and tumor, both indicative of hypoxia. Nevertheless, many of these voxels had tumor-to-blood (TBR) values lower than the 1.4 level commonly expected for hypoxic tissues, possibly due to the low rate-constants for tracer flow between the vasculature and tumor cells in these voxels. Conclusions Time-courses of FMISO uptake in NSCLC tumor voxels are best analyzed using an irreversible two-tissue compartment model, fits of which provide more precise parameter values than those of a three-tissue model. Changes in fitted model parameter values indicate that levels of hypoxia rise with increasing distance from tumor edges. The average FMISO binding rate-constant is higher for voxels in tumor centers than in the next tumor layer out, but the average value of the more simplistic TBR metric is lower in tumor centers. For both metrics higher values might be considered indicative of hypoxia, and the mismatch in this case is likely to be due to poor perfusion at the tumor center. Kinetics analysis of dynamic PET images may therefore provide more accurate measures of the hypoxic status of such regions than the simpler TBR metric, a hypothesis we are presently exploring in a study of tumor imaging versus histopathology.

The identification of genetic determinants that underpin tumor radioresistance can help the development of targeted radiosensitizers or aid personalization of radiotherapy treatment. Here we identify signal recognition particle 72kDa (SRP72) as a novel gene involved in radioresistance. Knockdown of SRP72 resulted in significant radiosensitization of HeLa (cervical), PSN-1 (pancreatic), and T24 (bladder), BT-549 (breast) and MCF7 (breast) tumor lines as measured by colony formation assays. SRP72 depletion also resulted in the radiosensitization of normal lung fibroblast cell lines (HFL1 and MRC-5), demonstrating that the effect is not restricted to tumor cells. Increased radiosensitivity was not due to impaired DNA damage signaling or repair as assessed by γ-H2AX foci formation. Instead SRP72 depletion was associated with elevated levels of apoptosis after irradiation, as measured by caspase 3/7 activity, PARP-cleavage and Annexin-V staining, and with an induction of the unfolded protein response. Together, our results show that SRP72 is a novel gene involved in radioresistance.

To investigate whether psychophysical, morphological, and/or optical characteristics of symptomatic non-advanced cataract are complementary to, or more appropriate than, visual acuity (VA) for the purposes of recording visual data that reflect subjective visual difficulty in patients with cataract that exhibit relative sparing of high contrast acuity (0.4 logarithm of minimal angle of resolution (logMAR) scale or better). Methods Eighty-two patients with symptomatic non-advanced cataract and no other ocular pathology were asked to complete a validated questionnaire, and to perform a series of visual function assessments including: corrected distance VA (CDVA); photopic and mesopic contrast sensitivity; photopic and mesopic glare disability (GD); reading acuity and reading speed; stereoacuity; and retinal sensitivity. Optical and morphological characteristics of the cataract were evaluated by lens optical density and by the Lens Opacities Classification System III, respectively. Correlations between questionnaire score and each of these measures were calculated. Results Statistically significant negative correlations were observed between the Rasch-scaled questionnaire score and mesopic GD (at 3 and 6 cycles per degree (cpd); r ¼ 0.396 (Po0.01) and 0.451 (Po0.05), respectively) and between the Rasch-scaled questionnaire score and photopic GD (at 3 and 6 cpd; r ¼ 0.328 (Po0.01) and 0.440 (Po0.01), respectively). Conclusion Symptomatic non-advanced cataract, in the presence of good CDVA, is associated with measurable subjective visual difficulty, best reflected in a decrease in mesopic and photopic GD (at medium spatial frequencies). CDVA does not reflect the patient’s visual dissatisfaction in such cases.

Charge-shift electrophoresis and isopycnic centrifugation were used to investigate the binding of detergents by B-cell and plasma cell immunoglobulin. Plasma cell immunoglobulin did not bind detergent; neither its electrophoretic migration nor buoyant density were altered by the presence of detergents. In contrast, B-cell immunoglobulin bound detergent, which resulted in a change in electrophoretic behavior and a reduced buoyant density in CsCI gradients. At least 20–23 molecules of Triton X-100 were bound by each molecule of B-cell immunoglobulin. With respect to detergent binding, B-cell immunoglobulin was similar to other hydrophobic integral membrane proteins. Since detergent binding was unaffected by the extent of glycosylation of the molecule, the hydrophobicity of B-cell immunoglobulin was most likely due to the chemical nature of the polypeptide chain. The approximate molecular weight of the heavy chain of surface IgG is 77,000 which is larger than that of secreted IgG with molecular weight of 67,000. The Fc portion of the immunoglobulin molecule is proposed as the most probable location for the site of detergent binding.

Background Pseudomonas aeruginosa airway infection is associated with a high mortality rate in cystic fibrosis. Lipopolysaccharide (LPS), a main constituent of the outer membrane of P. aeruginosa, is responsible for activation of innate immune response but its role on airway epithelium ion transport, is not well known. The aim of this study was to determine the role for P. aeruginosa LPS in modulating chloride secretion and intracellular calcium in the human bronchial epithelial cell line, 16HBE14o−. Methods We used intracellular calcium imaging and short-circuit current measurement upon exposure of cells to P. aeruginosa LPS. Results Apical LPS stimulated intracellular calcium release and calcium entry and enhanced chloride secretion. This latter effect was significantly inhibited by CFTR(inh)-172 and BAPTA-AM (intracellular Ca 2+ chelator). Conclusions Our data provides evidence for a new role of P. aeruginosa LPS in stimulating calcium entry and release and a subsequent chloride secretion via CFTR in human bronchial epithelium.

Many clinically effective chemotherapeutic agents exert their toxicity through formation of DNA interstrand cross-links (ICL). These prevent the replication and transcription of DNA and if unrepaired lead to cell death. Despite the success of ICL inducing agents in treating cancers, issues remain with a lack of sensitivity in some tumours, therapy-limiting toxicity and acquired resistance and tumour recurrence. The modulation of ICL repair could improve chemotherapy outcomes and the specific targeting of ICL repair in quiescent cells may selectively target cancer stem cells and ameliorate issues with recurrence. This thesis seeks to identify potential druggable targets that may sensitise cancer cells to ICL inducing agents and elucidate the role of ICL repair in quiescence. Previous studies have suggested ICL repair occurs chiefly in S-phase when DNA replication forks collide with ICLs. However, recent research in budding yeast, using Xenopus cell-free extracts and using genetic reporter assays in mammalian cells suggests that ICL repair can occur in the G1 phase of the cell cycle via the nucleotide excision repair (NER) and translesional synthesis (TLS) pathways. Previously this G1 pathway had not been well explored in mammalian cells. In this thesis a model for G1 ICL repair was created using a mammalian cell line. Flow cytometry analysis (FACS) of cell cycle dynamics following ICL induction in phase G1 demonstrated that repair of ICL can occur in G1 and this appears time dependent. Further analysis by FACS, comet assays and cell survival analysis confirmed repair of ICL in G1 and suggested that the NER factors XPF, XPB and XPD and the exonucleases SNM1A and EXO1 may be required for ICL repair in G1 but not the XPG, FAN1 or CSB repair proteins. Along-side these studies, a large-scale siRNA screen of 640 DNA damage repair genes was performed in a lung cancer cell line utilising colony formation assays to identify potential cisplatin and radiotherapy sensitising genes. Following a secondary screen, eight potential radiation and cisplatin sensitising genes were identified and examined in further detail, with the gene PIF1 finally confirmed in CRISPR/Cas9 knockout cells as a potential cisplatin sensitising gene. The findings in this thesis confirm ICL repair can occur in G1 in mammalian cells and identify PIF1 as a potential cisplatin sensitiser in human cells. In the future, targeting of the specific endonucleases responsible for G1 ICL repair or PIF1 may offer a potential clinical cancer treatment option.

Radiotherapy is one of the pillars of modern cancer therapy. However, side effects associated with normal tissue toxicity limit the effectiveness of this treatment. It is therefore desirable to develop tumour-specific radiosensitising agents, which can increase tumour cell kill without increasing the radiosensitivity of the surrounding normal tissue. This project aimed at validating the radiosensitising potential of depletion of the histone acetyltransferase KAT7, and elucidating the underlying mechanism of action. My results showed a radiosensitising effect of KAT7 depletion in seven tumour cell lines. At the same time, a panel of normal tissue cell lines was not radiosensitised. The depletion of KAT7 caused general cell cycle changes, most notably a twofold increase in the proportion of mitotic cells. At the same time, the G2M checkpoint governing entry into mitosis appeared to be impaired. Livecell imaging confirmed a more than threefold increase in mitotic catastrophe following irradiation in KAT7-depleted cells. An oligonucleotide microarray also showed the downregulation of key members of the mitotic pathway, and patient data demonstrated higher expression of KAT7 to be associated with worse prognosis. In summary, this project supports the tumour-specific radiosensitising potential of KAT7 depletion. This novel target for radiosensitisation seems to exert its effects through altering entry into and progression through mitosis.

Radiotherapy is second only to surgery in the curative management of patients with cancer, and yet the molecular mechanisms that determine the sensitivity of tumours to radiation remain largely unclear. A high-throughput radiosensitivity screening method based on clonogenicity was developed and a siRNA library against kinase targets was screened. The gold standard colony formation endpoint was chosen for determining reproductive cell death after radiation treatment, since effects on proliferation often do not reflect survival. Thiamine pyrophosphokinase-1 (TPK1), a key component of Vitamin B1/thiamine metabolism, was identified as a target for radiosensitisation. TPK1 knockdown caused significant radiosensitisation in cancer but not normal tissue cell lines. Other means of blocking this pathway such as knockdown of thiamine transporter-1 (THTR1) or treatment with the thiamine analogue pyrithiamine hydrobromide (PyrH) caused significant tumour specific radiosensitisation. There was persistent DNA damage in cells irradiated after TPK1 and THTR1 knockdown or PyrH treatment. Thus this screen allowed the identification of thiamine metabolism as a novel radiosensitisation target that affects DNA repair. Short-term modulation of thiamine metabolism could be a clinically exploitable strategy to achieve tumour specific radiosensitisation. Three additional genes, signal recognition particle-72 kDa (SRP72), glycogen synthase 3-beta (GSK3β) and MAP/Microtubule Affinity-Regulating Kinase 2 (MARK2) were also investigated. Knockdown of these genes radiosensitised both tumour and normal tissue cell lines and expression of two of them, GSK3β and SRP72 were found to be associated with poor recurrence-free survival in early breast cancer patients.