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Two types of tests are reported in this thesis: (1) tests of push -out specimens and (2) tests of composite cellular T -beams. The components of the composite members tested were short lengths of steel I -beams and concrete ribbed slabs. The ribbed slabs were formed by the inclusion of cellular sheet steel decking. Various rib sizes were used by varying the cell height and cell width of the decking. The components of a specimen were tied together by stud shear connectors. The tests were made to investigate the influence of cell geometry on the behaviour of composite cellular members and to provide a rational approach to their design. / Thesis / Master of Engineering (MEngr)

Osteosarcoma is the most common primary malignancy of bone. 5-year survival has remained stable at around 60-70% for 40 years. However, a number of patients will suffer from recurrent and/or metastatic disease representing a large unmet clinical need. CSPG4 is a transmembrane protein which is expressed on a number of progenitor cells and tumour types. Preliminary work had found CSPG4 present in osteosarcoma tumour samples. In this study, CSPG4 mRNA and protein expression was demonstrated in clinical samples and model cell lines. CSPG4 mRNA is overexpressed in osteosarcoma samples compared to mature osteoblast cells, the putative cell of origin for osteosarcoma. In a cohort of patients, CSPG4 protein expression was found on 86% of samples. Furthermore, CSPG4 expression was demonstrated in U2OS, MG63, HOS, HOS-MNNG and 143B osteosarcoma cell lines. CSPG4 protein expression was successfully deleted in 143B cells using CRISPR/Cas9 technology. Two stable CSPG4-negative cell lines were produced. CSPG4 expression was then reintroduced into negative cell lines, as well as the parental 143B cell line. This created a panel of 6 cell lines with differing CSPG4 expression. Furthermore, siRNA treatment of U2OS, MG63, 143B and U87MG cell lines reduced CSPG4 expression. These cells provided another panel with varying CSPG4 expression for in vitro investigation. In vitro experiments failed to demonstrate a role for CSPG4 in osteosarcoma tumorigenesis. The CRISPR/Cas9 cell panel found that CSPG4 expression did not influence cell proliferation, adhesion and spreading on fibronectin or collagen-I, cell migration, chemosensitivity or anchorage-independent growth. Similarly, the siRNA cell panel found that CSPG4 expression did not influence cell proliferation or anchorage-independent growth. In vivo experimentation did not demonstrate a role for CSPG4 in mediating osteosarcoma tumour growth or metastatic spread. Treatment with a sc-Fv antibody fragment failed to demonstrate specific toxicity of CSPG4-positive cell lines. These results indicate that CSPG4 plays no role in osteosarcoma tumour cell behaviour. However, due to its wide expression pattern it represents a viable therapeutic option for drug targeting.

The human protein kinase superfamily consists of over 500 members that individually control specific aspects of cell behavior and collectively control the complete range of cellular processes. That such a large group of proteins is able to uniquely diversify and establish individual identities while retaining common enzymatic function and significant sequence/structural conservation is remarkable. The means by which this is achieved is poorly understood, and we have begun to examine the issue by performing a comparative analysis of the catalytic domain of protein kinases. A novel approach for protein structural alignment has revealed a high degree of similarity found across the kinase superfamily, with variability confined largely to a single region thought to be involved in substrate binding. The similarity detected is not limited to amino acids, but includes a group of conserved water molecules that play important structural roles in stabilizing critical residues and the fold of the kinase domain. The development of a novel technique for identifying kinase substrates on a large scale directly from cell lysate has revealed that substrate specificity is not what discriminates the closely related p38α and β mitogen-activated protein kinases. Instead cellular localization appears to be their distinguishing characteristic, at least during myoblast differentiation. Together these results highlight the extent of conservation, as well as the minimal variability, that is found in the catalytic domain of all protein kinase superfamily members, and that while distantly related kinases may be distinguished by substrate specificity, closely related kinases are likely to be distinguished by other factors. Although these results focus on representative members of the kinase superfamily, they give insight as to how all protein kinases likely diversified and established unique non-redundant identities. In addition, the novel techniques developed and presented here for structural alignment and substrate discovery offer new tools for studying molecular biology and cell signaling.

The human protein kinase superfamily consists of over 500 members that individually control specific aspects of cell behavior and collectively control the complete range of cellular processes. That such a large group of proteins is able to uniquely diversify and establish individual identities while retaining common enzymatic function and significant sequence/structural conservation is remarkable. The means by which this is achieved is poorly understood, and we have begun to examine the issue by performing a comparative analysis of the catalytic domain of protein kinases. A novel approach for protein structural alignment has revealed a high degree of similarity found across the kinase superfamily, with variability confined largely to a single region thought to be involved in substrate binding. The similarity detected is not limited to amino acids, but includes a group of conserved water molecules that play important structural roles in stabilizing critical residues and the fold of the kinase domain. The development of a novel technique for identifying kinase substrates on a large scale directly from cell lysate has revealed that substrate specificity is not what discriminates the closely related p38α and β mitogen-activated protein kinases. Instead cellular localization appears to be their distinguishing characteristic, at least during myoblast differentiation. Together these results highlight the extent of conservation, as well as the minimal variability, that is found in the catalytic domain of all protein kinase superfamily members, and that while distantly related kinases may be distinguished by substrate specificity, closely related kinases are likely to be distinguished by other factors. Although these results focus on representative members of the kinase superfamily, they give insight as to how all protein kinases likely diversified and established unique non-redundant identities. In addition, the novel techniques developed and presented here for structural alignment and substrate discovery offer new tools for studying molecular biology and cell signaling.

The human protein kinase superfamily consists of over 500 members that individually control specific aspects of cell behavior and collectively control the complete range of cellular processes. That such a large group of proteins is able to uniquely diversify and establish individual identities while retaining common enzymatic function and significant sequence/structural conservation is remarkable. The means by which this is achieved is poorly understood, and we have begun to examine the issue by performing a comparative analysis of the catalytic domain of protein kinases. A novel approach for protein structural alignment has revealed a high degree of similarity found across the kinase superfamily, with variability confined largely to a single region thought to be involved in substrate binding. The similarity detected is not limited to amino acids, but includes a group of conserved water molecules that play important structural roles in stabilizing critical residues and the fold of the kinase domain. The development of a novel technique for identifying kinase substrates on a large scale directly from cell lysate has revealed that substrate specificity is not what discriminates the closely related p38α and β mitogen-activated protein kinases. Instead cellular localization appears to be their distinguishing characteristic, at least during myoblast differentiation. Together these results highlight the extent of conservation, as well as the minimal variability, that is found in the catalytic domain of all protein kinase superfamily members, and that while distantly related kinases may be distinguished by substrate specificity, closely related kinases are likely to be distinguished by other factors. Although these results focus on representative members of the kinase superfamily, they give insight as to how all protein kinases likely diversified and established unique non-redundant identities. In addition, the novel techniques developed and presented here for structural alignment and substrate discovery offer new tools for studying molecular biology and cell signaling.