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    Development and applications of filamentous phage-derived particles in immunotherapy and diagnostics : a dissertation presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University (Manawatū), New Zealand
    (Massey University, 2020) Rajič, Marina
    Most vaccines that are currently in clinical use induce antibody-mediated responses. However, for many infectious diseases, T cells are an essential part of naturally acquired protective immune responses. T cell-inducing vaccines, such as the one developed in this research, could additionally be used for treatments in cancer or chronic viral infections. One way to target the immune cells and stimulate their responses is to use filamentous phage particles. Filamentous phages (Ff) are ssDNA viruses that infect Escherichia coli, which have been adapted and used extensively in phage display technology and nanotechnology. In this research, a filamentous phage (Ff)-based vaccine carrier was constructed to allow the tuneable display of non-protein immune adjuvant molecules (BODIPY-α-GalCer) and antigenic peptides (OVA; MHC I + MHC II) on the same particle. For the first time, azide groups were incorporated into the recombinant pVIII phage coat proteins that expressed recombinant peptides. Azide groups were subsequently used to attach fluorescently labelled adjuvant molecules, which were successfully presented to NKT cells in vivo. Additionally, high induction of in vitro proliferation of OVA-specific CD8+ T-cells was achieved. However, the Ff-derived particle use outside the laboratory is hindered because they are genetically modified viruses, which in addition, carry antibiotic resistance genes that can be horizontally transferred to gut bacteria. These limitations were overcome by developing a system for efficient production of non-replicating, controllable-length protein-DNA nanorods, derived from Ff, named BSFnano (~100 × 6 nm). In this research, functionalised BSFnano particles were constructed, and their application in diagnostics was tested in a proof of concept dipstick assay for detection of a soluble analyte (fibronectin). For the first time, an ultrasensitive dipstick assay was achieved with Ff-derived nanorods, detecting the test-analyte at a concentration of as low as 0.04 pg/μL, equivalent to only 100,000 molecules/μL. Overall, while the phage-based vaccine produced in this research elicited CD8+ T-cell responses in vitro, but not in vivo, the Ff-derived nanorods were successfully functionalised and tested in lateral flow immunoassay, with promising implications for use in point of care diagnostics.
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    A portable multi-modal micro-imaging system for automated scanning and image stitching applications : a thesis submitted to Massey University in accordance with the requirements of the degree of Master of Engineering in the School of Engineering and Advanced Technology
    (Massey University, 2019) Naqvi, Adam
    Microscopic imaging is an important element in many fields like biology, medicine, diagnostics, engineering, and materials research. Muti-modal microscopes are ideal for imaging samples that reveal unseen structures that could not otherwise be seen with normal bright-field microscopes. Point-of-care micro-imaging devices are ones that can deliver the features of a microscope in areas where access to a laboratory or medical facilities are scarce. This thesis presents the development of a portable micro-imaging system that uses multi-modal illumination to image samples in bright-field, fluorescence, ambient and laser diffraction modes. A systematic design method has been used to develop the system from the conceptual phase to a working prototype. The system incorporates variable magnification through an inverted turret system and a GUI application for live image view, automatic scanning, auto-focusing and image processing. The utility of the system is demonstrated through imaging stained biological samples for a local industry application. The acquired images are measured against sharpness and noise. It is observed that the sharpness and noise of the images produced vary with the type of sample: samples with higher contrast generally produce sharper images with less noise. It has also been found that diffused ambient illumination produces the most consistent sharpness and noise scores between magnifications. Performance of algorithms used is discussed and improvements are suggested for building a more compact and stable platform including a method to calibrate measurements for particle size estimation.