PhD, EngD and MSc by research theses (Cranfield Health)

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Browsing PhD, EngD and MSc by research theses (Cranfield Health) by Supervisor "Bossi, Alessandra"

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    Discovery and quantification of proteins of biological relevance through differential proteomics and biosensing
    (Cranfield University, 2012-04) Lonardoni, Francesco; Bossi, Alessandra; Chianella, Iva
    Medical diagnosis is the process of attempting to determine and/or identify a possible disease or disorder. This process is revealed by biomarkers, defined by The Food and Drug Administration (FDA) as “characteristics that are objectively measured and evaluated as indicators of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention”. The process of biomarker discovery has been boosted in the last years by proteomics, a research discipline that takes a snapshot of the entire wealth of proteins in an organism/ tissue/ cell/ body fluid. An implementation of the analysis methods can help in isolate proteins present in the low range of concentrations, such as biomarkers very often are. An established biomarker can further be measured with the help of biosensors, devices that can be employed in the point-of care diagnostics. This PhD thesis shows and discusses the results of three projects in the field of protein biomarkers discovery and quantification. The first project exploited proteomics techniques to find relevant protein markers for Intrauterine Growth Restriction (IUGR) in cordonal blood serum (UCS) and amniotic fluid (AF). A 14 proteins in UCS and 11 in AF were successfully identified and found to be differentially expressed. Molecularly Imprinted Polymers (MIPs) directed towards proteins and peptides containing phosphotyrosine were then produced, with the final goal of selectively extracting phosphopeptides from a peptide mixture. An alteration of the phosphorylation pattern is in fact often associated to important diseases such as cancer. The polymers were produced as nanoparticles, that were characterised with Dynamic Light Scattering (DLS) and Atomic Force Microscopy (AFM). A recipe was also tested for binding capacity towards phosphotyrosine. A Surface Plasmon Resonance (SPR) biosensor to quantify hepcidin hormone was finally produced. This is the major subject in iron homeostasis in vertebrates and marker of iron unbalance diseases. A calibration curve was made and affinity/kinetic parameters for the ligand employed were measured.
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    Molecularly imprinted polymers for protome analysis
    (Cranfield University, 2008-01) Bonini, Francesca; Piletsky, Sergey A.; Bossi, Alessandra
    Fast and efficient methods for the detection of insurgence and progression of diseases are at the basis of modern diagnostics and medicine. In this concern, biomarkers represent a powerful diagnostic tool, as their expression profiles well correlate with the pathology progression. Thus, the pathological state could be diagnosed by measuring the altered presence of a biomarker. In this direction, conspicuous help has been given by proteomics, intended as the study of the protein pattern of a sample and most frequently performed by two-dimensional electrophoresis. Although the proteome approach is a powerful analytical method, its application to biological samples for the detection and quantification of putative biomarkers is hampered by technical problems, in fact, the wide diversity in concentrations exhibited by the proteins present in the biological samples, with a concentration range spanning over nine orders of magnitude, and the relative abundance of each protein, are responsible of masking the less abundant species and of their loss in traceability. The aim of my PhD project is to apply Molecularly Imprinted Technology to the specific removal of a high abundance protein (Human Serum Albumin, HSA) frequently affecting proteomic analysis, in order to increase the detection of potential biomarkers. This technology allows the creation of artificial recognition sites in synthetic polymers for a specific protein. These sites are tailor-made in situ by co-polymerisation of functional monomers and cross-linkers around the template molecules. Two different approaches have been assayed in order to remove HSA: • Immobilisation of protein template on a rigid silica support (bead) and creation of polymer around beads. • Polymerisation in bulk of a polymer with protein template and application of this polymer to multicompartment electrolyser. In both of the cases, the chemical and structural features of the polymers have been analysed, after that they have been applied to complex proteome pre-treatment, obtaining encouraging results.