Experimental study of the mechanical properties of light gauge cold-formed steels at elevated temperatures

In recent times, light gauge cold-formed steel sections have been used extensively as primary load bearing structural members in many applications in the building industry. Fire safety design of structures using such sections has therefore become more important. Deterioration of mechanical properties of yield stress and elasticity modulus is considered the most important factor affecting the performance of steel structures in fires. Hence there is a need to fully understand the mechanical properties of light gauge cold-formed steels at elevated temperatures. A research project based on experimental studies was therefore undertaken to investigate the deterioration of mechanical properties of light gauge cold-formed steels. Tensile coupon tests were undertaken to determine the mechanical properties of these steels made of both low and high strength steels and thicknesses of 0.60, 0.80 and 0.95 mm at temperatures ranging from 20 to 800ºC. Test results showed that the currently available reduction factors are unsafe to use in the fire safety design of cold-formed steel structures. Therefore new predictive equations were developed for the mechanical properties of yield strength and elasticity modulus at elevated temperatures. This paper presents the details of the experimental study, and the results including the developed equations. It also includes details of a stress-strain model for light gauge cold-formed steels at elevated temperatures.

Distortional buckling tests of cold-formed steel compression members at elevated temperatures

In recent times, light gauge cold-formed steel sections have been used extensively since they have a very high strength to weight ratio compared with thicker hot-rolled steel sections. However, they are susceptible to various buckling modes including a distortional mode and hence show complex behaviour under fire conditions. Therefore a research project based on detailed experimental studies was undertaken to investigate the distortional buckling behaviour of light gauge cold-formed steel compression members under simulated fire conditions. More than 150 axial compression tests were undertaken at uniform ambient and elevated temperatures. Two types of cross sections were selected with nominal thicknesses of 0.60, 0.80, and 0.95 mm. Both low (G250) and high (G550) strength steels were used. Distortional buckling tests were conducted at six different temperatures in the range of 20 to 800°C. The ultimate loads of compression members subject to distortional buckling were then used to review the adequacy of the current design rules at ambient and elevated temperatures. This paper presents the details of this experimental study and the results.

Recombinant protein production using a Tobacco yellow dwarf virus-based episomal expression vector : control of Rep activity

Over the past decade, plants have been used as expression hosts for the production of pharmaceutically important and commercially valuable proteins. Plants offer many advantages over other expression systems such as lower production costs, rapid scale up of production, similar post-translational modification as animals and the low likelihood of contamination with animal pathogens, microbial toxins or oncogenic sequences. However, improving recombinant protein yield remains one of the greatest challenges to molecular farming. In-Plant Activation (InPAct) is a newly developed technology that offers activatable and high-level expression of heterologous proteins in plants. InPAct vectors contain the geminivirus cis elements essential for rolling circle replication (RCR) and are arranged such that the gene of interest is only expressed in the presence of the cognate viral replication-associated protein (Rep). The expression of Rep in planta may be controlled by a tissue-specific, developmentally regulated or chemically inducible promoter such that heterologous protein accumulation can be spatially and temporally controlled. One of the challenges for the successful exploitation of InPAct technology is the control of Rep expression as even very low levels of this protein can reduce transformation efficiency, cause abnormal phenotypes and premature activation of the InPAct vector in regenerated plants. Tight regulation over transgene expression is also essential if expressing cytotoxic products. Unfortunately, many tissue-specific and inducible promoters are unsuitable for controlling expression of Rep due to low basal activity in the absence of inducer or in tissues other than the target tissue. This PhD aimed to control Rep activity through the production of single chain variable fragments (scFvs) specific to the motif III of Tobacco yellow dwarf virus (TbYDV) Rep. Due to the important role played by the conserved motif III in the RCR, it was postulated that such scFvs can be used to neutralise the activity of the low amount of Rep expressed from a “leaky” inducible promoter, thus preventing activation of the TbYDV-based InPAct vector until intentional induction. Such scFvs could also offer the potential to confer partial or complete resistance to TbYDV, and possibly heterologous viruses as motif III is conserved between geminiviruses. Studies were first undertaken to determine the levels of TbYDV Rep and TbYDV replication-associated protein A (RepA) required for optimal transgene expression from a TbYDV-based InPAct vector. Transient assays in a non-regenerable Nicotiana tabacum (NT-1) cell line were undertaken using a TbYDV-based InPAct vector containing the uidA reporter gene (encoding GUS) in combination with TbYDV Rep and RepA under the control of promoters with high (CaMV 35S) or low (Banana bunchy top virus DNA-R, BT1) activity. The replication enhancer protein of Tomato leaf curl begomovirus (ToLCV), REn, was also used in some co-bombardment experiments to examine whether RepA could be substituted by a replication enhancer from another geminivirus genus. GUS expression was observed both quantitatively and qualitatively by fluorometric and histochemical assays, respectively. GUS expression from the TbYDV-based InPAct vector was found to be greater when Rep was expected to be expressed at low levels (BT1 promoter) rather than high levels (35S promoter). GUS expression was further enhanced when Rep and RepA were co-bombarded with a low ratio of Rep to RepA. Substituting TbYDV RepA with ToLCV REn also enhanced GUS expression but more importantly highest GUS expression was observed when cells were co-transformed with expression vectors directing low levels of Rep and high levels of RepA irrespective of the level of REn. In this case, GUS expression was approximately 74-fold higher than that from a non-replicating vector. The use of different terminators, namely CaMV 35S and Nos terminators, in InPAct vectors was found to influence GUS expression. In the presence of Rep, GUS expression was greater using pInPActGUS-Nos rather than pInPActGUS-35S. The only instance of GUS expression being greater from vectors containing the 35S terminator was when comparing expression from cells transformed with Rep, RepA and REnexpressing vectors and either non-replicating vectors, p35SGS-Nos or p35SGS-35S. This difference was most likely caused by an interaction of viral replication proteins with each other and the terminators. These results indicated that (i) the level of replication associated proteins is critical to high transgene expression, (ii) the choice of terminator within the InPAct vector may affect expression levels and (iii) very low levels of Rep can activate InPAct vectors hence controlling its activity is critical. Prior to generating recombinant scFvs, a recombinant TbYDV Rep was produced in E. coli to act as a control to enable the screening for Rep-specific antibodies. A bacterial expression vector was constructed to express recombinant TbYDV Rep with an Nterminal His-tag (N-His-Rep). Despite investigating several purification techniques including Ni-NTA, anion exchange, hydrophobic interaction and size exclusion chromatography, N-His-Rep could only be partially purified using a Ni-NTA column under native conditions. Although it was not certain that this recombinant N-His-Rep had the same conformation as the native TbYDV Rep and was functional, results from an electromobility shift assay (EMSA) showed that N-His-Rep was able to interact with the TbYDV LIR and was, therefore, possibly functional. Two hybridoma cell lines from mice, immunised with a synthetic peptide containing the TbYDV Rep motif III amino acid sequence, were generated by GenScript (USA). Monoclonal antibodies secreted by the two hybridoma cell lines were first screened against denatured N-His-Rep in Western analysis. After demonstrating their ability to bind N-His-Rep, two scFvs (scFv1 and scFv2) were generated using a PCR-based approach. Whereas the variable heavy chain (VH) from both cell lines could be amplified, only the variable light chain (VL) from cell line 2 was amplified. As a result, scFv1 contained VH and VL from cell line 1, whereas scFv2 contained VH from cell line 2 and VL from cell line 1. Both scFvs were first expressed in E. coli in order to evaluate their affinity to the recombinant TbYDV N-His-Rep. The preliminary results demonstrated that both scFvs were able to bind to the denatured N-His-Rep. However, EMSAs revealed that only scFv2 was able to bind to native N-His-Rep and prevent it from interacting with the TbYDV LIR. Each scFv was cloned into plant expression vectors and co-bombarded into NT-1 cells with the TbYDV-based InPAct GUS expression vector and pBT1-Rep to examine whether the scFvs could prevent Rep from mediating RCR. Although it was expected that the addition of the scFvs would result in decreased GUS expression, GUS expression was found to slightly increase. This increase was even more pronounced when the scFvs were targeted to the cell nucleus by the inclusion of the Simian virus 40 large T antigen (SV40) nuclear localisation signal (NLS). It was postulated that the scFvs were binding to a proportion of Rep, leaving a small amount available to mediate RCR. The outcomes of this project provide evidence that very high levels of recombinant protein can theoretically be expressed using InPAct vectors with judicious selection and control of viral replication proteins. However, the question of whether the scFvs generated in this project have sufficient affinity for TbYDV Rep to prevent its activity in a stably transformed plant remains unknown. It may be that other scFvs with different combinations of VH and VL may have greater affinity for TbYDV Rep. Such scFvs, when expressed at high levels in planta, might also confer resistance to TbYDV and possibly heterologous geminiviruses.

Protein complementation assay as a display system for screening protein libraries in the intracellular environment

A wide range of screening strategies have been employed to isolate antibodies and other proteins with specific attributes, including binding affinity, specificity, stability and improved expression. However, there remains no high-throughput system to screen for target-binding proteins in a mammalian, intracellular environment. Such a system would allow binding reagents to be isolated against intracellular clinical targets such as cell signalling proteins associated with tumour formation (p53, ras, cyclin E), proteins associated with neurodegenerative disorders (huntingtin, betaamyloid precursor protein), and various proteins crucial to viral replication (e.g. HIV-1 proteins such as Tat, Rev and Vif-1), which are difficult to screen by phage, ribosome or cell-surface display. This study used the β-lactamase protein complementation assay (PCA) as the display and selection component of a system for screening a protein library in the cytoplasm of HEK 293T cells. The colicin E7 (ColE7) and Immunity protein 7 (Imm7) *Escherichia coli* proteins were used as model interaction partners for developing the system. These proteins drove effective β-lactamase complementation, resulting in a signal-to-noise ratio (9:1 – 13:1) comparable to that of other β-lactamase PCAs described in the literature. The model Imm7-ColE7 interaction was then used to validate protocols for library screening. Single positive cells that harboured the Imm7 and ColE7 binding partners were identified and isolated using flow cytometric cell sorting in combination with the fluorescent β-lactamase substrate, CCF2/AM. A single-cell PCR was then used to amplify the Imm7 coding sequence directly from each sorted cell. With the screening system validated, it was then used to screen a protein library based the Imm7 scaffold against a proof-of-principle target. The wild-type Imm7 sequence, as well as mutants with wild-type residues in the ColE7- binding loop were enriched from the library after a single round of selection, which is consistent with other eukaryotic screening systems such as yeast and mammalian cell-surface display. In summary, this thesis describes a new technology for screening protein libraries in a mammalian, intracellular environment. This system has the potential to complement existing screening technologies by allowing access to intracellular proteins and expanding the range of targets available to the pharmaceutical industry.

Prevalence of Staphylococcus aureus strains in an Australian cohort, 1989-2003 : evidence for the low prevalence of the toxic shock toxin and Panton-Valentine leukocidin genes

The purpose of this paper is to determine the prevalence of the toxic shock toxin gene (tst) and to enumerate the circulating strains of methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) in Australian isolates collected over two decades. The aim was to subtype these strains using the binary genes pvl, cna, sdrE, pUB110 and pT181. Isolates were assayed using real-time polymerase chain reaction (PCR) for mecA, nuc, 16 S rRNA, eight single-nucleotide polymorphisms (SNPs) and for five binary genes. Two realtime PCR assays were developed for tst. The 90 MRSA isolates belonged to CC239 (39 in 1989, 38 in 1996 and ten in 2003), CC1 (two in 2003) and CC22 (one in 2003). The majority of the 210 MSSA isolates belonged to CC1 (26), CC5 (24) and CC78 (23). Only 18 isolates were tst-positive and only 15 were pvl-positive. Nine MSSA isolates belonged to five binary types of ST93, including two pvlpositive types. The proportion of tst-positive and pvl-positive isolates was low and no significant increase was demonstrated. Dominant MSSA clonal complexes were similar to those seen elsewhere, with the exception of CC78. CC239 MRSA (AUS-2/3) was the predominant MRSA but decreased significantly in prevalence, while CC22 (EMRSA-15) and CC1 (WA-1) emerged. Genetically diverse ST93 MSSA predated the emergence of ST93- MRSA (the Queensland clone).

Adiabatic compression testing - part I : historical development and evaluation of fluid dynamic processes including shock-wave considerations

Adiabatic compression testing of components in gaseous oxygen is a test method that is utilized worldwide and is commonly required to qualify a component for ignition tolerance under its intended service. This testing is required by many industry standards organizations and government agencies. This paper traces the background of adiabatic compression testing in the oxygen community and discusses the thermodynamic and fluid dynamic processes that occur during rapid pressure surges. This paper is the first of several papers by the authors on the subject of adiabatic compression testing and is presented as a non-comprehensive background and introduction.

Bioactive mesopore-glass microspheres with controllable protein-delivery properties by biomimetic surface modification

Microsphere systems with the ideal properties for bone regeneration need to be bioactive, and at the same time possess the capacity for controlled protein/drug-delivery; however, the current crop of microsphere system fails to fulfill these properties. The aim of this study was to develop a novel protein-delivery system of bioactive mesoporous glass (MBG) microspheres by a biomimetic method through controlling the density of apatite on the surface of microspheres, for potential bone tissue regeneration. MBG microspheres were prepared by using the method of alginate cross-linking with Ca2+ ions. The cellular bioactivity of MBG microspheres was evaluated by investigating the proliferation and attachment of bone marrow stromal cell (BMSC). The loading efficiency and release kinetics of bovine serum albumin (BSA) on MBG microspheres were investigated after coprecipitating with biomimetic apatite in simulated body fluids (SBF). The results showed that MBG microspheres supported BMSC attachment and the Si containing ionic products from MBG microspheres stimulated BMSCs proliferation. The density of apatite on MBG microspheres increased with the length of soaking time in SBF. BSA-loading efficiency of MBG was significantly enhanced by co-precipitating with apatite. Furthermore, the loading efficiency and release kinetics of BSA could be controlled by controlling the density of apatite formed on MBG microspheres. Our results suggest that MBG microspheres are a promising protein-delivery system as a filling material for bone defect healing and regeneration.

Porous PLGA microspheres effectively loaded with BSA protein by electrospraying combined with phase separation in liquid nitrogen

Polymer microspheres loaded with bioactive particles, biomolecules, proteins, and/or growth factors play important roles in tissue engineering, drug delivery, and cell therapy. The conventional double emulsion method and a new method of electrospraying into liquid nitrogen were used to prepare bovine serum albumin (BAS)-loaded poly(lactic-co-glycolic acid) (PLGA) porous microspheres. The particle size, the surface morphology and the internal porous structure of the microspheres were observed using scanning electron microscopy (SEM). The loading efficiency, the encapsulation efficiency, and the release profile of the BSA-loaded PLGA microspheres were measured and studied. It was shown that the microspheres from double emulsion had smaller particle sizes (3-50 m), a less porous structure, a poor loading efficiency (5.2 %), and a poor encapsulation efficiency (43.5%). However, the microspheres from the electrospraying into liquid nitrogen had larger particle sizes (400-600 m), a highly porous structure, a high loading efficiency (12.2%), and a high encapsulation efficiency (93.8%). Thus the combination of electrospraying with freezing in liquid nitrogen and subsequent freeze drying represented a suitable way to produce polymer microspheres for effective loading and sustained release of proteins.

Lateral-torsional buckling of cold-formed steel beams at ambient temperature

Cold-formed steel beams are increasingly used as floor joists and bearers in residential, industrial and commercial buildings. Their structural behaviour and moment capacities are influenced by lateral-torsional buckling and hence a research study was undertaken to investigate the lateral-torsional buckling behaviour of cold-formed steel lipped channel beams at ambient and elevated temperatures. For this purpose a finite element model of a simply supported cold-formed steel lipped channel beam under uniform bending was developed first and validated using available numberical and experimental results. It was then used in a detailed parametric study to simulate the lateral-torsional behaviour of cold-formed steel beams under varying conditions. The moment capacity results were then compared with the predictions from the current ambient temperature design rules in Australia, New Zealand, American and European codes for cold-formed steel structures. Some very interesting results have been obtained. European design rules are found to be conservative while Australian and American design rules are unsafe. This paper presents the results of finite element analyses for ambient temperature conditions, and the comparison with the current design rules.

Structural behaviour and design of cold-formed steel beams at elevated temperatures

Cold-formed steel members are extensively used in the building construction industry, especially in residential, commercial and industrial buildings. In recent times, fire safety has become important in structural design due to increased fire damage to properties and loss of lives. However, past research into the fire performance of cold-formed steel members has been limited, and was confined to compression members. Therefore a research project was undertaken to investigate the structural behaviour of compact cold-formed steel lipped channel beams subject to inelastic local buckling and yielding, and lateral-torsional buckling effects under simulated fire conditions and associated section and member moment capacities. In the first phase of this research, an experimental study based on tensile coupon tests was undertaken to obtain the mechanical properties of elastic modulus and yield strength and the stress-strain relationship of cold-formed steels at uniform ambient and elevated temperatures up to 700oC. The mechanical properties deteriorated with increasing temperature and are likely to reduce the strength of cold-formed beams under fire conditions. Predictive equations were developed for yield strength and elastic modulus reduction factors while a modification was proposed for the stressstrain model at elevated temperatures. These results were used in the numerical modelling phases investigating the section and member moment capacities. The second phase of this research involved the development and validation of two finite element models to simulate the behaviour of compact cold-formed steel lipped channel beams subject to local buckling and yielding, and lateral-torsional buckling effects. Both models were first validated for elastic buckling. Lateral-torsional buckling tests of compact lipped channel beams were conducted at ambient temperature in order to validate the finite element model in predicting the non-linear ultimate strength behaviour. The results from this experimental study did not agree well with those from the developed experimental finite element model due to some unavoidable problems with testing. However, it highlighted the importance of magnitude and direction of initial geometric imperfection as well as the failure direction, and thus led to further enhancement of the finite element model. The finite element model for lateral-torsional buckling was then validated using the available experimental and numerical ultimate moment capacity results from past research. The third phase based on the validated finite element models included detailed parametric studies of section and member moment capacities of compact lipped channel beams at ambient temperature, and provided the basis for similar studies at elevated temperatures. The results showed the existence of inelastic reserve capacity for compact cold-formed steel beams at ambient temperature. However, full plastic capacity was not achieved by the mono-symmetric cold-formed steel beams. Suitable recommendations were made in relation to the accuracy and suitability of current design rules for section moment capacity. Comparison of member capacity results from finite element analyses with current design rules showed that they do not give accurate predictions of lateral-torsional buckling capacities at ambient temperature and hence new design rules were developed. The fourth phase of this research investigated the section and member moment capacities of compact lipped channel beams at uniform elevated temperatures based on detailed parametric studies using the validated finite element models. The results showed the existence of inelastic reserve capacity at elevated temperatures. Suitable recommendations were made in relation to the accuracy and suitability of current design rules for section moment capacity in fire design codes, ambient temperature design codes as well as those proposed by other researchers. The results showed that lateral-torsional buckling capacities are dependent on the ratio of yield strength and elasticity modulus reduction factors and the level of non-linearity in the stress-strain curves at elevated temperatures in addition to the temperature. Current design rules do not include the effects of non-linear stress-strain relationship and therefore their predictions were found to be inaccurate. Therefore a new design rule that uses a nonlinearity factor, which is defined as the ratio of the limit of proportionality to the yield stress at a given temperature, was developed for cold-formed steel beams subject to lateral-torsional buckling at elevated temperatures. This thesis presents the details and results of the experimental and numerical studies conducted in this research including a comparison of results with predictions using available design rules. It also presents the recommendations made regarding the accuracy of current design rules as well as the new developed design rules for coldformed steel beams both at ambient and elevated temperatures.

Immunogenicity and protective efficacy of an anti-Streptococcus pyogenes vaccine candidate in multiple animal species

Streptococcus pyogenes, also known as Group A Streptococcus (GAS) has been associated with a range of diseases from the mild pharyngitis and pyoderma to more severe invasive infections such as streptococcal toxic shock. GAS also causes a number of non-suppurative post-infectious diseases such as rheumatic fever, rheumatic heart disease and glomerulonephritis. The large extent of GAS disease burden necessitates the need for a prophylactic vaccine that could target the diverse GAS emm types circulating globally. Anti-GAS vaccine strategies have focused primarily on the GAS M-protein, an extracellular virulence factor anchored to GAS cell wall. As opposed to the hypervariable N-terminal region, the C-terminal portion of the protein is highly conserved among different GAS emm types and is the focus of a leading GAS vaccine candidate, J8-DT/alum. The vaccine candidate J8-DT/alum was shown to be immunogenic in mice, rabbits and the non-human primates, hamadryas baboons. Similar responses to J8-DT/alum were observed after subcutaneous and intramuscular immunization with J8-DT/alum, in mice and in rabbits. Further assessment of parameters that may influence the immunogenicity of J8-DT demonstrated that the immune responses were identical in male and female mice and the use of alum as an adjuvant in the vaccine formulation significantly increased its immunogenicity, resulting in a long-lived serum IgG response. Contrary to the previous findings, the data in this thesis indicates that a primary immunization with J8-DT/alum (50ƒÊg) followed by a single boost is sufficient to generate a robust immune response in mice. As expected, the IgG response to J8- DT/alum was a Th2 type response consisting predominantly of the isotype IgG1 accompanied by lower levels of IgG2a. Intramuscular vaccination of rabbits with J8-DT/alum demonstrated that an increase in the dose of J8-DT/alum up to 500ƒÊg does not have an impact on the serum IgG titers achieved. Similar to the immune response in mice, immunization with J8-DT/alum in baboons also established that a 60ƒÊg dose compared to either 30ƒÊg or 120ƒÊg was sufficient to generate a robust immune response. Interestingly, mucosal infection of naive baboons with a M1 GAS strain did not induce a J8-specific serum IgG response. As J8-DT/alum mediated protection has been previously reported to be due to the J8- specific antibody formed, the efficacy of J8-DT antibodies was determined in vitro and in vivo. In vitro opsonization and in vivo passive transfer confirmed the protective potential of J8-DT antibodies. A reduction in the bacterial burden after challenge with a bioluminescent M49 GAS strain in mice that were passively administered J8-DT IgG established that protection due to J8-DT was mediated by antibodies. The GAS burden in infected mice was monitored using bioluminescent imaging in addition to traditional CFU assays. Bioluminescent GAS strains including the ‘rheumatogenic’ M1 GAS could not be generated due to limitations with transformation of GAS, however, a M49 GAS strain was utilized during BLI. The M49 serotype is traditionally a ‘nephritogenic’ serotype associated with post-streptococcal glomerulonephritis. Anti- J8-DT antibodies now have been shown to be protective against multiple GAS strains such as M49 and M1. This study evaluated the immunogenicity of J8-DT/alum in different species of experimental animals in preparation for phase I human clinical trials and provided the ground work for the development of a rapid non-invasive assay for evaluation of vaccine candidates.