Abstract Lysyl hydroxylases (E.C. 1.14.11.4, LHs) have three isoenzymes that are found in humans and mice, and they hydroxylate lysine residues in collagens and other proteins containing collagenous sequences. The hydroxylysines formed are crucial for the intermolecular collagen crosslinks that stabilise collagen fibres, thereby providing the stiffness and stability required by various tissues. In addition, hydroxylysines serve as attachment sites for carbohydrates, whose functions on collagen molecules are not completely understood yet. In humans, lack of LH1 causes Ehlers-Danlos syndrome (EDS) VIA, which is characterised, for example, by severe progressive kyphoscoliosis and muscular hypotonia with joint laxity. Mutations in the LH2 gene are associated with Bruck syndrome, which is characterised by fragile bones with congenital joint contractures. In the present work recombinant human lysyl hydroxylases were produced in insect cells and purified to homogeneity. Limited proteolysis revealed that LHs consist of at least three structural domains. The N-terminal domain plays no role in the lysyl hydroxylase activity, but instead, is responsible for the recently reported glucosyltransferase activity of LH3, and the galactosyltransferase activity reported here for the first time. The LH polypeptide lacking the N-terminal domain is a fully active LH with Km values identical to those of full-length enzyme. In addition, direct evidence is shown that LH2, but not LH1 or LH3, hydroxylates the telopeptide lysine residues of fibrillar collagens. All three recombinant LHs were able to hydroxylate the synthetic peptides representing the helical hydroxylation sites in types I and IV collagens, with some differences in the Vmax and Km values. In addition, all three LHs hydroxylated the collagenous domain of coexpressed type I procollagen chain to similar extend. In this study mouse lines lacking LH3 or LH1 were created and analysed. Unexpectedly, the LH3 null mice died during the embryonal period due to fragmentation of basement membranes. Type IV collagen, one of the major components in basement membranes, aggregates on its way to extracellular space and is absent from the basement membranes making them fragile. This is most probably caused by abnormal processing of type IV collagen due to decreased glucosyltransferase activity of the LH3 null embryos. The first mouse model for human EDS VIA is presented here. The LH1 null mice did not have kyphoscoliosis characteristic of EDS VIA, but showed gait abnormalities due to muscular hypotonia and possible joint laxity, as also seen in EDS VIA patients. In addition, the null mice died occasionally from aortic ruptures. Ultra structural analysis revealed degradation of smooth muscle cells and abnormal collagen fibres even in non-ruptured aortas of LH1 null mice. The hydroxylation of lysine residues and crosslinking in LH1 null mice were also abnormal, as in human EDS VIA patients. The LH1 null mouse line provides an excellent tool for analysing several aspects of human EDS VIA, including muscular hypotonia, abnormalities in collagen fibres and their crosslinking.

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<p>Interstrand cross-linking (ICL) damage to DNA is cytotoxic as it blocks replication and transcription. This cytotoxicity is exploited in anti-cancer therapies, but increased ICL repair limits the efficacy of these chemotherapies. SNM1A (sensitive to nitrogen mustard 1A), of the beta-CASP family of nucleases, has been shown to participate in the initiation of one of the ICL repair processes. Biochemical studies of SNM1A have been limited due to insolubility and instability of SNM1A in bacteria and insect cell lines and toxicity in human cell lines. Work reported in this thesis describes a novel and efficient method of generating active protein from inclusion body expression of the beta-CASP domain of SNM1A. This refolded beta-CASP domain shows 5’ exonuclease activity on single stranded and double stranded DNA in vitro. Nevertheless, this domain alone is unable to complement <em>pso2</em> null ICL repair defects in<em> S. cerevisiae</em> after exposure to ICL agents. These functional studies of the beta-CASP domain of SNM1A will be helpful in directing future research on its role in ICL repair. Additionally, this will aid future structural and inhibitor studies of this essential interstrand cross-link repair protein, SNM1A.</p> / Master of Science (MSc)

The influence of silane treated amorphous fumed silica fillers on properties of the cured epoxy was examined in the first part of the study. Silica particles were treated with 3- aminopropyldiethoxymethylsilane (APDS) and 3-aminopropyltriethoxysilane (APTS) coupling agents. The filler and coupling agents decreased the mobility of the polymer chains in the vicinity of the filler leading to an increase in the activation energy for the glass transition and an increase in cooperativity. Fumed silica did not significantly affect moisture diffusion properties. Next, a linear dilatometer was used to investigate the effects of cure conditions, mold types, and the presence of filler in the model epoxy. These studies revealed that there was substantial shrinkage in the cured epoxy on heating it through its glass transition region. The shrinkage was determined to be the result of stress in the epoxy generated during cure and could be minimized by curing at lower temperatures, followed by a postcuring heat treatment. Additional free volume in the sample increased the magnitude of the shrinkage by allowing increased stress release through increased network mobility. Decreasing the polymer mobility by adding fillers decreased the observed shrinkage. The influence of the model epoxy crosslink density was examined by varying the content of 1,4-butanediol in the model system. Addition of 1,4-butanediol led to a decrease in the modulus and glass transition temperature, which resulted in a reduction in residual stress and subsequent shrinkage. Moisture uptake increased with the addition of 1,4-butanediol due to an increase in the free volume of the epoxy. However, even with greater moisture uptake, the addition of 1,4-butanediol to the epoxy increased its adhesion to quartz by promoting lower residual stress and increased energy dissipation. These results indicate that bulk diffusion of water is not the controlling factor in adhesive degradation in this system. / Ph. D.