Development of monoclonal antibodies for the identification of novel invasion associated targets in human cancer.

Monoclonal antibodies (MAbs) have emerged as an important therapeutic modality for the treatment of cancer, due to their high specificity, low toxicity, and the ability to activate components of the immune system. The research carried out in this thesis aims to identify novel antigens associated with cancer invasion, that could form the basis of anti-invasive therapeutic targets, through the generation of MAbs directed against the highly invasive MiaPaCa-2 clone 3 pancreatic cell line, and the MDA-MB-435-SF breast cancer cell line. Two MAbs were identified that could successfully block cancer invasion in vitro. MAb 7B7 G5 (2) significantly reduced invasion in the MiaPaCa-2 clone 3 pancreatic cell line; the SKBR-3 and MDA-MB-231 breast cancer cell lines; the DLKP-M and H1299 lung cancer cell lines; the SNB-19 glioma cell line and the HCT-116 colon cancer cell line. Inhibition of invasion was also observed in the Lox IMVI melanoma cell line, but not significantly so. This MAb also significantly decreased cell motility in the MiaPaCa-2 clone 3 cell line. MAb 9E1 24 (6) significantly decreased cell invasion in the MiaPaCa-2 clone 3, MDA-MB-231, DLKP-I, DLKP-M, H1299, C/68 and Lox IMVI cell lines. Invasion was also inhibited in the SKBR-3 cell line, but not significantly so. Surprisingly, invasion was increased in the HCT-116 colon cancer cell line, following incubation with this MAb. Other invasion-related processes were also decreased following incubation with the MAb 9E1 24 (6) and 7B7 G5 (2); MiaPaCa-2 clone 3 adhesion to fibronectin, and MMP-9 activity in the MDA-MB-231 breast cancer cell line. Immunohistochemical analysis of 9E1 24 (6) revealed that its target antigen is expressed to varying degrees in a wide range of tumour types (colon adenocarcinoma, pancreatic, breast, B-Cell lymphoma, Retinoblastoma and Glioma). Weaker staining was observed in normal colon, liver and prostate tissues. MAb 9E1 24 (6) was shown to react with a 75kDa protein band on Western blot analysis. Immunoprecipitation studies, followed by LC-MS/MS analysis, revealed that its target antigen was Annexin A6, a 75kDa cellular calcium and phospholipid binding protein. This was further corroborated by decreased expression of the reactive 9E1 24 (6) band in Annexin A6-silenced cells. siRNA silencing of Annexin A6 significantly reduced invasion in the MiaPaCa-2 clone 3 and DLKP-M cell lines, suggesting a role for this protein in the invasion process. A cross-linked immunoprecipitation approach with MAb 7B7 G5(2) revealed two bands at approximately 70 and 80kDa. LC-MS/MS analysis identified these as Ku70 and Ku80 respectively, two subunits of the Ku heterodimer, which is involved in DNA double strand break repair. siRNA silencing of these two subunits in the MiaPaCa-2 clone 3 and DLKP-M cell lines significantly reduced levels of invasion and motility, indicating that they play a role in both processes. Immunofluorescence analysis on Ku70 and Ku80 silenced MiaPaCa-2 clone 3 cells revealed a significant decrease in MAb 7B7 G5 (2) reactivity on Ku80, but not Ku70, silenced cells, suggesting that the Ku80 subunit is the main target antigen for MAb 7B7 G5 (2). The research presented in this thesis is proof of principle of how MAbs can be successfully generated that specifically target invasion-related proteins, and can block cancer invasion in vitro. The identified proteins may have the potential to become useful therapeutic targets for the treatment of invasive cancers, and could lead to the development of new drugs that specifically target metastatic cancer cells

Improving the quality of the personalized electronic program guide

As Digital TV subscribers are offered more and more channels, it is becoming increasingly difficult for them to locate the right programme information at the right time. The personalized Electronic Programme Guide (pEPG) is one solution to this problem; it leverages artificial intelligence and user profiling techniques to learn about the viewing preferences of individual users in order to compile personalized viewing guides that fit their individual preferences. Very often the limited availability of profiling information is a key limiting factor in such personalized recommender systems. For example, it is well known that collaborative filtering approaches suffer significantly from the sparsity problem, which exists because the expected item-overlap between profiles is usually very low. In this article we address the sparsity problem in the Digital TV domain. We propose the use of data mining techniques as a way of supplementing meagre ratings-based profile knowledge with additional item-similarity knowledge that can be automatically discovered by mining user profiles. We argue that this new similarity knowledge can significantly enhance the performance of a recommender system in even the sparsest of profile spaces. Moreover, we provide an extensive evaluation of our approach using two large-scale, state-of-the-art online systems—PTVPlus, a personalized TV listings portal and Físchlár, an online digital video library system

The prevalence of severe sepsis or septic shock in an Irish emergency department

Severe sepsis and septic shock are among the leading causes of death globally. Despite the central role the emergency department (ED) plays in the early identification of patients presenting to hospital with sepsis, the prevalence of severe sepsis and septic shock in the Irish ED setting has not been described. The primary aim of this study was to measure the prevalence of severe sepsis or septic shock in an Irish adult ED setting. The clinical records of patients presenting to the ED over a four-week period were retrospectively reviewed to determine if they met the current Health Service Executive (HSE) criteria for severe sepsis or septic shock. Overall, 3,585 adult patients attended the ED during the study period, with 42 patients meeting the criteria for severe sepsis or septic shock. The ED prevalence of severe sepsis or septic shock was 11.7 patients (95% CI 8.1 – 15.4%) per 1000 ED attendances

Industry 4.0 driven statistical analysis of investment casting process demonstrates the value of digitalisation

The purpose of this research is to perform statistical data analysis of currently manually collected data in an area of the industrial manufacturing organisation employed in this study that is not digitalised to show the value that can be achieved through digitalisation. The insights gained through analysis of the data can be used to drive decision making in relation to the optimisation of input parameters to minimise the level of defective parts. The parts under investigation in this study were ceramic shells used in the manufacturing process of orthopaedic metal implants. The ceramic shell is a crucial element in the investment casting process because molten metal is poured into the ceramic shell to form the shape of the metal orthopaedic implant. Hence, by minimising the number of defective ceramic shells, there are fewer defective metal implants produced, resulting in cost savings and increased efficiency of the manufacturing process. A number of scientific questions to establish the relationship between the quantity of scrapped products and the level of the silica component in the ceramic slurry were defined and a series of independent t-tests were conducted to address these questions. The results from the t-tests showed the statistically optimal percentage of silica in the binder of the ceramic slurry to minimise the rate of a particular scrap type caused by thin or weak areas of the shell. These results demonstrate the value of analysing digital data relating to the manufacturing process to understand relationships between parameters in the manufacturing process and effectively root-cause scrap outputs. The results from the analysis gave rise to the implementation of a digitalised data collection system that allows continuous monitoring of the components in the ceramic slurry to ensure they are in the optimal specified range. Hence, the quality and yield rate of the orthopaedic implants are maintained at a high level. The digital data collection system also acts as a resource containing historical data for further potential scrap root-cause analysis

Polymorphisms near TBX5 and GDF7 are associated with increased risk for Barrett's esophagus.

BACKGROUND & AIMS: Barrett's esophagus (BE) increases the risk of esophageal adenocarcinoma (EAC). We found the risk to be BE has been associated with single nucleotide polymorphisms (SNPs) on chromosome 6p21 (within the HLA region) and on 16q23, where the closest protein-coding gene is FOXF1. Subsequently, the Barrett's and Esophageal Adenocarcinoma Consortium (BEACON) identified risk loci for BE and esophageal adenocarcinoma near CRTC1 and BARX1, and within 100 kb of FOXP1. We aimed to identify further SNPs that increased BE risk and to validate previously reported associations. METHODS: We performed a genome-wide association study (GWAS) to identify variants associated with BE and further analyzed promising variants identified by BEACON by genotyping 10,158 patients with BE and 21,062 controls. RESULTS: We identified 2 SNPs not previously associated with BE: rs3072 (2p24.1; odds ratio [OR] = 1.14; 95% CI: 1.09-1.18; P = 1.8 × 10(-11)) and rs2701108 (12q24.21; OR = 0.90; 95% CI: 0.86-0.93; P = 7.5 × 10(-9)). The closest protein-coding genes were respectively GDF7 (rs3072), which encodes a ligand in the bone morphogenetic protein pathway, and TBX5 (rs2701108), which encodes a transcription factor that regulates esophageal and cardiac development. Our data also supported in BE cases 3 risk SNPs identified by BEACON (rs2687201, rs11789015, and rs10423674). Meta-analysis of all data identified another SNP associated with BE and esophageal adenocarcinoma: rs3784262, within ALDH1A2 (OR = 0.90; 95% CI: 0.87-0.93; P = 3.72 × 10(-9)). CONCLUSIONS: We identified 2 loci associated with risk of BE and provided data to support a further locus. The genes we found to be associated with risk for BE encode transcription factors involved in thoracic, diaphragmatic, and esophageal development or proteins involved in the inflammatory response