Selective up-regulation of the mRNA of LOXL4, a member of the LOX matrix amine oxidase family, significantly correlated with lymph node metastases and higher tumour stages in head and neck squamous cell carcinomas (HNSCC). To evaluate the diagnostic and prognostic value of the protein we produced an antibody specific for LOXL4 and assessed the expression in 317 human HNSCC specimens. The LOXL4 protein was detected in 92.7% of primary tumours, in 97.8% of lymph node metastases and in affected oral mucosa with high-grade dysplasia, but was absent in various non-neoplastic tissues of the head and neck. TNM categories and overall survival did not link to grades of immunoreactivity. Studies in cultured primary hypopharyngeal HTB-43 carcinoma cells detected perinuclear and cell surface expression of LOXL4, but no nuclear localisation. Therefore, its interactive SRCR-domains and catalytic activity combined with tumour cell specific expression and cell surface associated location indicate multiple functions in tumour cell adhesion and interactions with the extracellular matrix. Our data suggest that LOXL4 is useful both as tumour marker and target in the treatment of HNSCC.

IPF is a progressive disease, characterized by dysregulated fibrosis of the extracellular matrix (ECM). The pathobiology of the disease is still unknown, and the median survival post-diagnosis is about 3-5 years. The two current US FDA approved drugs for IPF (nintedanib and pirfenidone) slow, but fail to reverse, disease progression. There is cumulating research that suggests the ECM is an active player in fibrosis. In this thesis, we summarized the current knowledge of ECM-cell interactions in the context of pulmonary fibrosis. To gain more mechanistic insight into the ECM characteristics that dictate cell behavior, we established a 3D ECM ex vivo system to assess the nonfibrotic and fibrotic ECM’s effect on fibroblasts. The ECM appears to promote both pathological and physiological cellular changes, depending on its structural and compositional properties. We also used this 3D ex vivo system as a preclinical tool to test the effect of directly inhibiting mechanotransduction in the fibrotic ECM – fibroblast profibrotic relationship. Lastly, since the fibrotic ECM seems to play a key role in progressive fibrosis, we evaluate if researchers are appropriately using the bleomycin model by starting interventions after ECM fibrosis is established. Over the past decade in the field, there has been an overall improvement in the appropriate therapeutic timing. In the preventative studies, however, there is still an inadequate characterization of inflammation. There is also poor transparency of preclinical-bleomycin data for clinically tested interventions for IPF. Addressing these shortcomings may improve the utility of the model at predicting an intervention’s success in clinical trials. These findings illustrate the ECM’s role in driving pulmonary fibrosis. Therefore, the ECM should be further investigated to understand disease progression, and reproduced in preclinical models to test interventions. This will improve the transition of pathobiological findings into efficient drug development for this devastating disease. / Thesis / Candidate in Philosophy / Idiopathic pulmonary fibrosis (IPF) (idiopathic - unknown cause; pulmonary - lungs; fibrosis - scarring) is characterized by progressive scarring of the lung extracellular matrix (ECM). The ECM is an organ’s backbone that provides structural and biochemical support to surrounding cells. Continued ECM scarring can lead to difficulty breathing, cough, and ultimately death. The cause of IPF is unknown, however, studies suggest that the scarred ECM can promote further scarring, and cause disease progression. In this thesis, we summarized the current knowledge of how the ECM interacts with cells. Using a 3D model we see that depending on the ECM’s structure and composition, it can promote both disease and healthy cellular changes. Lastly, we evaluate if researchers are appropriately using the bleomycin model (most common preclinical model for pulmonary fibrosis) by testing interventions after ECM fibrosis is established. We propose changes to improve its usefulness as a preclinical tool for IPF.

Cardiovascular disease is a leading cause of death worldwide. Changes in the cardiac extracellular matrix (ECM) are associated with cardiac pathologies such as cardiomyopathy and cardiac hypertrophy. The ECM is a dynamic scaffold of proteoglycans, fibrous proteins, and glycoproteins that sheathes and protects many organs and tissues, including the heart, by attenuating mechanical stress. Misregulation of ECM proteins triggers changes in matrix stiffness, which can lead to age-associated and congenital heart defects. ECM rigidity is also important to the migration of cells, such as hemocytes, the invertebrate blood cells. In the embryo, hemocytes also perform fibroblast functions, through the deposition of the ECM proteins Collagen and Laminin. Hemocytes are hypothesized to be critical for ECM assembly, and by extension, for heart development. The consequences of impaired hemocyte function in the embryo and during larval growth are unknown and are the focus of this research. Using Drosophila melanogaster as a model, I used genetic tools to manipulate hemocyte survival and motility to assess their role in ECM organization and structure around the heart. Concerted gene knockdown and confocal microscopy techniques were employed to evaluate the effects of altered hemocyte abundance and motility on hemocyte behaviour and resulting changes to the ECM. Here I provide evidence to support a role for hemocytes in the turnover of a vital ECM protein, the Type IV Collagen Viking. I also developed a novel protocol to photobleach and observe fluorescence recovery in intact, living larvae using confocal microscopy. Recovery of fluorescence of GFP tagged ECM is a measure of the rate of ECM protein turnover during development or growth. This novel technique has allowed for assessment of recovery of Viking-GFP after photobleaching in vivo, as a measue of Viking protein turnover at the cardiac ECM. This new technique can be employed to determine the turnover of other major ECM proteins. Combining hemocyte impairment with photobleaching provides the opportunity to observe innate protein turnover at the ECM in real time, both in normal and hemocyte-deprived matrices. Recovery of Viking-GFP fluorescence was also observed in hemocyte-deprived conditions. My findings reveal gradual recovery of Viking-GFP at the cardiac ECM in controls, and potentially slower recovery in hemocyte-impaired conditions. These observations suggest a role of hemocytes in ECM protein turnover. This work will help reveal the role of hemocytes in organizing the cardiac ECM and provides a novel technique for the in vivo assessment of ECM protein turnover. Ultimately, this research sheds light on how hemocyte function affects overall structure of the cardiac ECM and contributes to an enhanced understanding of how changes in this ECM influence predisposition to and progress of cardiac disease. / Thesis / Master of Science (MSc)

In recent decades science has become an increasingly multidisciplinary field in which the lines that used to divide starkly different fields have blurred or disappeared completely. This work is a compendium of different angles focused at exploring disease progression of cancer biology through the perspective of mechanical engineering. We explore cancer through a holistic approach considering mechanistic, physical, genetic biology, biochemical, and immune cells to explore how the interplay with fiber networks can expand our understanding. We explored the physical interplay with biological processes of fibroblastic cells and show how these are critically regulated by forces that alter their ability to coil depends on fiber curvature and adhesion strength; thus, showing how cellular processes are driven by the balances of mechanical forces. Conversely, not all cell types are driven by the same factors, where we report that the structural features of migratory DCs enable them to be less influenced by the differences in fiber diameters, contrasting drastically what we previously reported on the other cell lines. Finally developing a novel composite nanofiber platform, we reported how some cancer cells are mechanistically influenced by the architecture of a substrate and thus resulting in completely different migratory responses that we have associated with key regulatory genes and responding completely differently when in the presence of clinically relevant molecular therapies. Overall, we investigated cancer biology through stiffness gradients, geometric influence through biophysics on myoblasts, and immune cell migration forces as a strong indicator of cell behavior. / Master of Science / Biology has historically been studied through chemistry and genetics, an approach that has produced incredible scientific discoveries such as vaccines and various therapies. Similarly, mechanical engineering has taken us to corners of the world that we never thought possible through the creation of machines, vehicles and the creation of new metal alloys. This research work is part of an emergent field of collaborative science which is paving the way to new ideas and the development of compound fields such as mechanobiology. Here we investigate how cells migrate through small rope-like environments that imitate the same fibers our cells can encounter in the body. We control the thickness, the arrangement, the orientation and the strength of these ropes to investigate how cells react to these environments, thus reporting on the new behaviors cells adopts in these conditions as well as their potential medical implications. Overall, we have developed new methods of studying cancer and other types of cells by tackling new questions using a mechanical perspective.

The chondroitin sulfate proteoglycan (CSPG) aggrecan forms link protein-stabilised complexes with hyaluronan (HA), via its N-terminal G1-domain, that provide cartilage with its load bearing properties. Similar aggregates (potentially containing new members of the link protein family), in which other CSPGs (i.e., versican, brevican and neurocan) substitute for aggrecan, may contribute to the structural integrity of many other tissues including skin and brain. In this thesis, cartilage link protein (cLP) and the G1-domains of aggrecan (AG1) and versican (VG1) were expressed in Drosophila S2 cells, purified to homogeneity and functionally characterised. The recombinant human proteins were found to have properties similar to those described for the native molecules. For example cLP formed dimers, and HA decasaccharides (HA 10-mers) were the minimum size that could compete effectively for their binding to polymeric HA. In addition, gel filtration and protein cross-linking/MALDI-TOF peptide fingerprinting showed that cLP and AG1 interact in the absence or presence of HA. Conversely, cLP and VG1 did not bind directly to each other hi solution yet formed ternary complexes with HA24. N-linked glycosylation of VG1 and AG1 was demonstrated to be unnecessary for either HA binding or the formation of ternary complexes. Additionally, the length of HA required to accommodate two G1-domains was found to be significantly larger for aggrecan than versican, which may reflect differences hi the conformation of HA stabilised on binding these proteins. To further investigate protein-HA interactions, fluorescent HA oligosaccharides were prepared and characterised. HA oligosaccharides labelled with the fluorophore 2-aminobenzoic acid (2AA) from four to 40 residues hi length were purified to homogeneity by ion exchange chromatography using a logarithmic gradient. Molecular weight and purity characterisation of HA oligosaccharides was facilitated by 2AA derivitisation since it enhanced signals in MALDI-TOF mass spectrometry and improves fluorophore-assisted carbohydrate electrophoresis (FACE) analysis by avoiding the inverted parabolic migration characteristic of 2-aminoacridone (AMAC) labelled sugars. The small size and shape of the fluorophore maintains the biological activity of the derivatised oligosaccharides, as demonstrated by their ability to compete for polymeric hyaluronan binding to VG1, AG1 and cLP. An electrophoretic mobility shift assay was used to study VG1 binding to 2AA-labelled HA 8-, 10-, 20-, 30- and 40-mers and although no stable VG1 binding was observed to labelled 8-mers, the equilibrium dissociation constant (100 nM) for VG1 with HA 10-mers was estimated from densitometry analysis of the free oligosaccharide. Interactions involving 2AA labelled HA 20-, 30-, and 40-mers with VG1 also displayed positive cooperativity. Therefore, oligosaccharides labelled with 2-aminobenzoic acid are biologically active and show excellent potential as probes in fluorescence-based assays that investigate protein-carbohydrate interactions.

published_or_final_version / Zoology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Surgery / Doctoral / Doctor of Philosophy

published_or_final_version / Dentistry / Master / Master of Orthodontics

published_or_final_version / abstract / Mechanical Engineering / Master / Master of Philosophy

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy