<p><strong>A compatible interface between a biomedical material and host tissue is paramount to the continual function and life-span of medical devices that reside in the body. However, the unfavourable host response that ensues when foreign materials inhabit the body must be overcome for sophisticated medical devices, such as artificial organs and real-time biosensors, to be used clinically. My thesis research commenced with a search to find a pharmaceutical compound that could be incorporated into a medical device to suppress the accumulation of fibrous tissue. A prolyl hydroxylase inhibitor, a drug developed to inhibit collagen synthesis, was found to be effective at inhibiting collagen deposition within and on the outer surface of a poly(lactic-glycolic acid) disc, and also limited connective tissue ingrowth. Furthermore, the drug suppressed Scavenger Receptor A (SRA) expression on a macrophage-like cell culture, a receptor known to contain a collagenous domain. The latter finding prompted a review of the literature, upon which it was discovered that SRA mediates leukocyte adhesion and binding to an assortment of materials, such as silica, modified polystyrene, titanium, and iron(III) oxide. As a result, a series of studies were initiated to investigate whether leukocytes use SRA to detect a range of different biomedical materials. Consequently, we found that SRA contributes very little to leukocyte binding of two common medical polymers, polystyrene and poly(lactic-co-glycolic acid), but may interact with the materials to affect the cytokine profile in the local environment. In a subsequent study, SRA was found to be crucial to the leukocyte binding of polyanionic hydrogels. In summary, we have identified a unique pharmaceutical strategy for suppressing the accumulation of fibrous tissue on medical devices in vivo, and uncovered a mechanism of leukocyte stimulation in response to incubation with biomedical materials that the material science research community was not previously aware of. </strong></p> / Doctor of Philosophy (PhD)

<p>Antisense oligonucleotides (ODN) may interfere in gene expression on the basis of hybridising to its complementary messenger RNA (mRNA) sequence in the cell thereby preventing the synthesis of the peptide. Therefore, these ODNs may be potential drugs to treat human diseases by “knocking down” the expression of responsible genes or correcting the maturation process of mRNA in the field called antisense therapy. Moreover, antisense ODNs upon labelling are also potential imaging agents to monitor gene expression <i>in vivo</i>, i.e. to accomplish <i>in vivo</i> hybridisation. This would provide a non-invasive tool compared to present methods, which require tissue samples. </p><p>This goal may be reached using positron emission tomography (PET) methodology. PET is a most advanced <i>in vivo</i> imaging technology, which would allow exploring the fate of radionuclide-labelled antisense ODNs in the body; thereby providing information about biodistribution and quantitative accumulation in tissues to assess pharmacokinetic properties of ODNs. This kind of evaluation is important as part of the characterisation of antisense therapeutics but also as part of the development of antisense imaging agents.</p><p>The present study aimed to investigate ⁷⁶Br- and ⁶⁸Ga-labelled ODNs of five different modifications: phosphodiester, phosphorothioate, 2'-<i>O</i>-methyl phosphodiester, locked nucleic acid (LNA), and peptide nucleic acid. The study included exploration of the hybridisation abilities of these ODNs after labelling; furthermore, the biodistribution, metabolite analysis and uptake of the ODNs in rats regarding non-hybridisation and hybridisation specific uptake was conducted. Among the ODNs studied, LNA-DNA mixmer (LNA and DNA nucleotides in alternation along the sequence) displayed the most promising characteristics considering a higher retention in tissues, stability and longer plasma residence. However, biodistribution data demonstrated a non-hybridisation specific distribution in rat tissues with kidney, liver, spleen and bone marrow being the organs of high uptake. Scavenger receptors or other saturable processes unrelated to hybridisation may play a role in tissue uptake and in clearance of antisense ODNs through these organs. These processes may be sequence dependent suggesting that proof of <i>in vivo</i> hybridisation through imaging needs much more elaborate evaluations than just comparison of sense and antisense sequences and proving dose-dependency.</p>

Antisense oligonucleotides (ODN) may interfere in gene expression on the basis of hybridising to its complementary messenger RNA (mRNA) sequence in the cell thereby preventing the synthesis of the peptide. Therefore, these ODNs may be potential drugs to treat human diseases by “knocking down” the expression of responsible genes or correcting the maturation process of mRNA in the field called antisense therapy. Moreover, antisense ODNs upon labelling are also potential imaging agents to monitor gene expression in vivo, i.e. to accomplish in vivo hybridisation. This would provide a non-invasive tool compared to present methods, which require tissue samples. This goal may be reached using positron emission tomography (PET) methodology. PET is a most advanced in vivo imaging technology, which would allow exploring the fate of radionuclide-labelled antisense ODNs in the body; thereby providing information about biodistribution and quantitative accumulation in tissues to assess pharmacokinetic properties of ODNs. This kind of evaluation is important as part of the characterisation of antisense therapeutics but also as part of the development of antisense imaging agents. The present study aimed to investigate 76Br- and 68Ga-labelled ODNs of five different modifications: phosphodiester, phosphorothioate, 2'-O-methyl phosphodiester, locked nucleic acid (LNA), and peptide nucleic acid. The study included exploration of the hybridisation abilities of these ODNs after labelling; furthermore, the biodistribution, metabolite analysis and uptake of the ODNs in rats regarding non-hybridisation and hybridisation specific uptake was conducted. Among the ODNs studied, LNA-DNA mixmer (LNA and DNA nucleotides in alternation along the sequence) displayed the most promising characteristics considering a higher retention in tissues, stability and longer plasma residence. However, biodistribution data demonstrated a non-hybridisation specific distribution in rat tissues with kidney, liver, spleen and bone marrow being the organs of high uptake. Scavenger receptors or other saturable processes unrelated to hybridisation may play a role in tissue uptake and in clearance of antisense ODNs through these organs. These processes may be sequence dependent suggesting that proof of in vivo hybridisation through imaging needs much more elaborate evaluations than just comparison of sense and antisense sequences and proving dose-dependency.

No description available.

<p>Class A scavenger receptors are multifunctional transmembrane glycoproteins that mediate macrophage functions like phagocytosis and endocytosis. The macrophage receptor with collagenous structure (MARCO) is one such receptor. It has been shown that the extracellular cysteine-rich domain of MARCO is responsible for ligand binding, but the role of the cytoplasmic domain in ligand uptake is unclear. The aim of the studies presented in this thesis is to characterize the role of the cytoplasmic domain of MARCO and to characterize the molecular pathway of MARCO-mediated endocytosis.</p> <p>Full-length human MARCO (hMARCO) and Δ1-34hMARCO, which lacks the first thirty-four amino acids were created in order to determine whether amino acids 1-34 contained residues required for receptor internalization and surface expression. The constructs were stably expressed in HEK293T cells and found to have similar levels of surface expression and same rate of internalization without ligand. Interestingly, hMARCO, but not Δ1-34hMARCO, surface expression was up-regulated upon ligand incubation.</p> <p>In order to ascertain the importance of clathrin, dynamin and actin in MARCO-mediated endocytosis, specific endocytic inhibitors were used. MARCO-mediated ligand uptake was inhibited when clathrin and actin polymerization and, dynamin functions were impaired by these inhibitors. Furthermore, ligand uptake by Δ1-34hMARCO-expressing HEK293T was insensitive to inhibitors of clthrin and dynamin but not inhibitors of actin.</p> <p>In conclusion, MARCO mediates endocytosis via a clathrin-mediated, dynamin-dependent pathway that involves actin. Amino acids 1-34, are required clathrin and dynamin but not actin functions during MARCO-mediated endocytosis. Additionally, amino acids 1-34 might also be important for MARCO recycling but not receptor internalization or surface expression.</p> / Master of Science (MSc)