Development and optimisation of photonic crystal based nanosensors

This thesis involved the development of two Biosensors and their associated assays for the detection of diseases, namely IBR and BVD for veterinary use and C1q protein as a biomarker to pancreatic cancer for medical application, using Surface Plasmon Resonance (SPR) and nanoplasmonics. SPR techniques have been used by a number of groups, both in research [1-3] and commercially [4, 5] , as a diagnostic tool for the detection of various biomolecules, especially antibodies [6-8]. The biosensor market is an ever expanding field, with new technology and new companies rapidly emerging on the market, for both human [8] and veterinary applications [9, 10]. In Chapter 2, we discuss the development of a simultaneous IBR and BVD virus assay for the detection of antibodies in bovine serum on an SPR-2 platform. Pancreatic cancer is the most lethal cancer by organ site, partially due to the lack of a reliable molecular signature for diagnostic testing. C1q protein has been recently proposed as a biomarker within a panel for the detection of pancreatic cancer. The third chapter discusses the fabrication, assays and characterisation of nanoplasmonic arrays. We will talk about developing C1q scFv antibody assays, clone screening of the antibodies and subsequently moving the assays onto the nanoplasmonic array platform for static assays, as well as a custom hybrid benchtop system as a diagnostic method for the detection of pancreatic cancer. Finally, in chapter 4, we move on to Guided Mode Resonance (GMR) sensors, as a low-cost option for potential use in Point-of Care diagnostics. C1q and BVD assays used in the prior formats are transferred to this platform, to ascertain its usability as a cost effective, reliable sensor for diagnostic testing. We discuss the fabrication, characterisation and assay development, as well as their use in the benchtop hybrid system.

THAWS: automated wireless sensor network development and deployment

This research focuses on the design and implementation of a tool to speed-up the development and deployment of heterogeneous wireless sensor networks. The THAWS (Tyndall Heterogeneous Automated Wireless Sensors) tool can be used to quickly create and configure application-specific sensor networks. THAWS presents the user with a choice of options, in order to characterise the desired functionality of the network. With this information, THAWS generates the necessary code from pre-written templates and well-tested, optimized software modules. This is then automatically compiled to form binary files for each node in the network. Wireless programming of the network completes the task of targeting the wireless network towards a specific sensing application. THAWS is an adaptable tool that works with both homogeneous and heterogeneous networks built from wireless sensor nodes that have been developed in the Tyndall National Institute.

Investigating the function of PINK1 in cancer development and pathogenesis

PTEN‐induced kinase 1 (PINK1) was identified initially in cancer cells as a gene up‐regulated by overexpression of the central tumour suppressor, PTEN. Loss‐of‐function mutations in PINK1 were discovered subsequently to cause autosomal recessive Parkinsonʹs disease (ARPD). Despite much research focusing on the proposed mechanism(s) through which loss of PINKI function causes neurodegeneration, few studies have focused on a direct role for this serine/threonine kinase in cancer biology. The focus of this thesis was to examine a direct role for PINK1 function in tumourigenesis. Initial studies showed that loss of PINK1 reduces tumour‐associated phenotypes including cell growth, colony formation and invasiveness, in several cell types in vitro, indicating a pro‐tumourigenic role for PINK1 in cancer. Furthermore, results revealed for the first time that PINK1 deletion, examined in mouse embryonic fibroblasts (MEFS) from PINK1 knock‐out animals, causes cell cycle defects, whereby cells arrest at in cytokinesis, giving rise to a highly significant increase in the number of multinucleated cells. This results in several key changes in the expression profile of cell cycle associated protein. In addition, PINK1‐deficient MEFs were found to resist cell cycle exit, with a proportion of cells remaining in proliferative phases upon removal of serum. The ability of cells to progress through mitosis conferred by PINK1 expression was independent of its kinase activity, while the cell cycle exit following serum withdrawal was kinase dependent. Investigations into the mechanism through which loss of PINK1 function gives rise to cell cycle defects revealed that dynamin related protein 1 (Drp1)‐mediated mitochondrial fission is enhanced in PINK1‐ deficient MEFs, and that increased expression of Drp1 on mitochondria and activation of Drp1 is highly significant in PINK1‐deficient multinucleated cells. Deregulated and increased levels and activation of mitochondrial fission via Drp1 was shown to be a major feature of cell cycle defects caused by PINK1 deletion, both during progression through G2/M and cell cycle exit following serum removal. Altered PINK1 localisation was also observed during progression of mitosis, and upon serum deprivation. Thus, PINK1 dissociated from the mitochondria during the mitotic phases and localised to mitochondria upon serum withdrawal. During serum withdrawal deletion of PINK1 disabled the ability of MEFs to increase mitochondrial membrane potential (ΔΨm), and increase autophagy. This was co‐incident with increased mitochondrial fission, and increased localisation of Drp1 to mitochondria following serum deprivation. Together, this indicates an inability of PINK1‐negative cells to respond protectively to this stress‐induced state, primarily via impaired mitochondrial function. In contrast, PINK1 overexpression was found to protect cells from DNA damage following treatment with oxidants. In addition, deletion of PINK1 blocked the ability of cells to re‐enter the cell cycle in response to insulin‐like growth factor‐1 (IGF‐1), a major cancer promoting agonistwhich acts primarily via PI3‐kinase/Akt activation. Furthermore, PINK1 mRNA expression was significantly increased following serum deprivation of MCF‐7 cells, and this was rendered more significant upon additional inhibition of PI3‐kinase. Conversely, IGF‐1 activation of PI3‐kinase/Akt causes a time‐dependent and significant reduction of PINK1 mRNA expression that was PI3‐kinase dependent. Together these results indicate that PINK1 expression is necessary for IGF‐1 signalling and is regulated reciprocally in the absence and presence of IGF‐1, via PI3‐kinase/Akt, a signalling system which has major tumour‐promoting capacity in cancer cell biology. The results of this thesis indicate PINK1 is a candidate tumour-promoting gene which has a significant function in the regulation of the cell cycle, and growth factor responses, at key cell cycle checkpoints, namely, during progression through G2/M and during exit of the cell cycle following removal of serum. Furthermore, the results reveal that the regulation of mitochondrial fission and Drp1 function is mechanistically important in the regulation of cell cycle control by PINK1. As deregulation of the cell cycle is linked to both tumourigenesis and neurodegeneration, the findings of this thesis are of importance not just for understanding cancer biology, but also in the context of PINK1‐associated neurodegeneration.

Self-neglect: development and evaluation of a self-neglect (SN-37) measurement instrument

Aim: To develop and evaluate the psychometric properties of an instrument for the measurement of self-neglect (SN).Conceptual Framework: An elder self-neglect (ESN) conceptual framework guided the literature review and scale development. The framework has two key dimensions physical/psycho-social and environmental and seven sub dimensions which are representative of the factors that can contribute to intentional and unintentional SN. Methods: A descriptive cross-sectional design was adopted to achieve the research aim. The study was conducted in two phases. Phase 1 involved the development of the questionnaire content and structure. Phase 2 focused on establishing the psychometric properties of the instrument. Content validity was established by a panel of 8 experts and piloted with 9 health and social care professionals. The instrument was subsequently posted with a stamped addressed envelope to 566 health and social care professionals who met specific eligibility criteria across the four HSE areas. A total of 341 questionnaires were returned, a response rate of 60% and 305 (50%) completed responses were included in exploratory factor analysis (EFA). Item and factor analyses were performed to elicit the instruments underlying factor structure and establish preliminary construct validity. Findings: Item and factor analyses resulted in a logically coherent, 37 items, five factor solution, explaining 55.6% of the cumulative variance. The factors were labelled: ‘Environment’, ‘Social Networks’, ‘Emotional and Behavioural Liability’, ‘Health Avoidance’ and ‘Self-Determinism’. The factor loadings were >0.40 for all items on each of the five subscales. Preliminary construct validity was supported by findings. Conclusion: The main outcome of this research is a 37 item Self-Neglect (SN-37) measurement instrument that was developed by EFA and underpinned by an ESN conceptual framework. Preliminary psychometric evaluation of the instrument is promising. Future work should be directed at establishing the construct and criterion related validity of the instrument.

Development and psychometric evaluation of the spirituality instrument (SpI-27) in a sample of people with chronic illness

Background: Spirituality is fundamental to all human beings, existing within a person, and developing until death. This research sought to operationalise spirituality in a sample of individuals with chronic illness. A review of the conceptual literature identified three dimensions of spirituality: connectedness, transcendence, and meaning in life. A review of the empirical literature identified one instrument that measures the three dimensions together. Yet, recent appraisals of this instrument highlighted issues with item formulation and limited evidence of reliability and validity. Aim: The aim of this research was to develop a theoretically-grounded instrument to measure spirituality – the Spirituality Instrument-27 (SpI-27). A secondary aim was to psychometrically evaluate this instrument in a sample of individuals with chronic illness (n=249). Methods: A two-phase design was adopted. Phase one consisted of the development of the SpI-27 based on item generation from a concept analysis, a literature review, and an instrument appraisal. The second phase established the psychometric properties of the instrument and included: a qualitative descriptive design to establish content validity; a pilot study to evaluate the mode of administration; and a descriptive correlational design to assess the instrument’s reliability and validity. Data were analysed using SPSS (Version 18). Results: Results of exploratory factor analysis concluded a final five-factor solution with 27 items. These five factors were labelled: Connectedness with Others, Self-Transcendence, Self-Cognisance, Conservationism, and Connectedness with a Higher Power. Cronbach’s alpha coefficients ranged from 0.823 to 0.911 for the five factors, and 0.904 for the overall scale, indicating high internal consistency. Paired-sample t-tests, intra-class correlations, and weighted kappa values supported the temporal stability of the instrument over 2 weeks. A significant positive correlation was found between the SpI-27 and the Spirituality Index of Well-Being, providing evidence for convergent validity. Conclusion: This research addresses a call for a theoretically-grounded instrument to measure spirituality.

Development and reliability of a direct access sensor using flip chip on flex technology with anisotropic conductive adhesive

Technological developments in biomedical microsystems are opening up new opportunities to improve healthcare procedures. Swallowable diagnostic sensing capsules are an example of these. In none of the diagnostic sensing capsules, is the sensor’s first level packaging achieved via Flip Chip Over Hole (FCOH) method using Anisotropic Conductive Adhesive (ACA). In a capsule application with direct access sensor (DAS), ACA not only provides the electrical interconnection but simultaneously seals the interconnect area and the underlying electronics. The development showed that the ACA FCOH was a viable option for the DAS interconnection. Adequate adhesive formed a strong joint that withstood a shear stress of 120N/mm2 and a compressive stress of 6N required to secure the final sensor assembly in place before encapsulation. Electrical characterization of the ACA joint in a fluid environment showed that the ACA was saturated with moisture and that the ions in the solution actively contributed to the leakage current, characterized by the varying rate of change of conductance. Long term hygrothermal aging of the ACA joint showed that a thermal strain of 0.004 and a hygroscopic strain of 0.0052 were present and resulted in a fatigue like process. In-vitro tests showed that high temperature and acidity had a deleterious effect of the ACA and its joint. It also showed that the ACA contact joints positioned at around or over 1mm would survive the gastrointestinal (GI) fluids and would be able to provide a reliable contact during the entire 72hr of the GI transit time. A final capsule demonstrator was achieved by successfully integrating the DAS, the battery and the final foldable circuitry into a glycerine capsule. Final capsule soak tests suggested that the silicone encapsulated system could survive the 72hr gut transition.

Functional educational characterisation of the utility of embalming fluids for anatomical education and research

Human cadavers have long been used to teach human anatomy and are increasingly used in other disciplines. Different embalming techniques have been reported in the literature; however there is no clear consensus on the opinion of anatomists on the utility of embalmed cadavers for the teaching of anatomy. To this end, we aimed to survey British and Irish anatomy teachers to report their opinions on different preservation methods for the teaching of anatomy. In this project eight human cadavers were embalmed using formalin, Genelyn, Thiel and Imperial College London- Soft Preserving (ICL-SP) techniques to compare different characteristics of these four techniques. The results of this thesis show that anatomy teachers consider hard-fixed cadavers not to be the most accurate teaching model in comparison to the human body, although it still serves as a useful teaching method (Chapter 2). In addition, our findings confirm that joints of cadavers embalmed using ICL-SP solution faithfully mimics joints of an unembalmed cadaver compared to the other techniques (Chapter 3). Embalming a human body prevents the deterioration in the quality of images and our findings highlight that the influence of the embalming solutions varied with the radiological modality used (Chapter 4). The method developed as part of this thesis enables anatomists and forensic scientists to quantify the decomposition rate of an embalmed human cadaver (Chapter 5). Formalin embalming solution showed the strongest antimicrobial abilities followed by Thiel, Genelyn and finally by ICL-SP (Chapter 6). The overarching viewpoint of this set of studies show that it is inaccurate to state that one embalming technique is ultimately the best. The value of each technique differs based on the requirement of the particular education or research area. Hence we highlight how different embalming techniques may be better suited to certain fields of study.