Aim: Subunit vaccines have received increasing attention due to their good safety profile. However, subunit vaccines feature low immunogenicity, and soluble antigen is largely ignored by the immune system due to its lack of danger signals. To stimulate an appropriate immune response, subunit antigen vaccines require the addition of an adjuvant and multiple administrations. This study aimed to formulate biodegradable lipid implants, containing a suitable adjuvant, which delivers antigen in a sustained manner. The physico-chemical characteristics of the implants and their ability to stimulate immune responses towards a model antigen in vivo were investigated. Methods: Lipid implants were prepared from phospholipid and cholesterol. Different adjuvants were added, and their potential to induce an immune response to the model antigen ovalbumin (OVA) was investigated. The adjuvants and immunomodulators assessed were Quil-A (QA), imiquimod, and an α-Galactosylceramide (α-GalCer) analogue. Liposomal dispersions were prepared using the lipid film hydration method. These were freeze-dried, and the powder compressed into matrices (diameter of 2 mm). Physico-chemical characterisation was undertaken by transmission electron microscopy (TEM) to investigate the release of colloidal structures (liposomes, immunostimulating complexes [ISCOMs]) upon hydration with release media. Surface changes of the implant matrices were analysed using scanning electron microscopy (SEM). The release of the fluorescently-labelled antigen ovalbumin (FITC-OVA) and its entrapment into the colloidal particles was investigated using spectrofluorophotometry. Additionally, incorporation of the cationic cholesterol derivative DC-cholesterol (DCCHOL) into implants to allow for charge-charge interactions with the negatively-charged OVA, and replacement of the phospholipid with a phospholipid having a higher transition temperature to facilitate the manufacturing process, were attempted and assessed. The immune response stimulated towards OVA released from the implants was analysed in vivo using a C57Bl/6 mouse model. Expansion of CD8⁺ T cells and CD8 T cells specific for the CD8 epitope of OVA (SIINFEKL), as well as expansion of CD4⁺ T cells, were assessed. The ability of implants to stimulate T cell proliferation and interferon-γ production after in vitro restimulation with OVA was analysed. Serum samples were analysed for OVA-specific IgG antibodies. Results: Lipid implants containing Quil-A released colloidal structures upon hydration with buffer. The type of colloids observed by TEM depended on the ratio of QA:cholesterol:phospholipid. Release of OVA was sustained over ten days in implants prepared with egg yolk PC. However, the release kinetics depended strongly on the choice of phospholipid. In vivo, lipid implants containing Quil-A evoked expansion of CD8⁺ T cells. The immune response to one implant was comparable to that obtained by two equivalent injection immunisations. Therefore, the implants obviated the need for multiple immunisations in the vaccination regime tested here. Expansion of CD8⁺ T cells towards the Quil-A-containing implant was greater than that achieved by the immunomodulators imiquimod and the α-GalCer analogue. Quil-A-containing implants produced OVA-specific IgG antibodies to a greater extent than the implants containing imiquimod or α-GalCer. Incorporation of the cationic DCCHOL did not increase the entrapment efficiency of OVA into liposomes. However, the in vivo investigation of DCCHOL-containirig implants showed an adjuvant effect of DCCHOL on antibody responses, but not on cell-mediated immunity. Conclusion: Lipid implants offer great potential as sustained release vaccine delivery systems. The lipid components in the implant formulation were well-tolerated and biodegradable. Lipid implants combine the advantages of sustained release of antigen and particulate delivery by the formation of colloidal particles.

The pressure-tension hypothesis is the governing dogma of orthodontic tooth movement. This theory proposes that the application of loads to the crown of a tooth during orthodontic mechano-therapy results in differential site-specific reactionary strains in the para-dental tissues. Briefly, following the application of orthodontic load the bone and periodontal ligament (PDL) on one side of the tooth is placed in compression favoring bone resorption, while on the other side of the tooth they are placed in tension favoring osteogenesis The present in vitro model provides a surrogate for the PDL on the tension side of the tooth during orthodontic tooth movement and aims to identify mechanically induced changes in the expression of osteo-regulatory cytokines in human PDL cell cultures in response to tensile mechanical strain. Materials and Methods: PDL explants were obtained from pathology free bicuspids of two human subjects following extraction of the teeth for orthodontic purposes. Following serial passage, cells were plated on Uniflex� plates and consigned to either the experimental or control groups. Experimental cells were exposed to a cyclic uniaxial tensile mechanical strain for 6,12 or 24 hours using the Flexercell FX 4000 strain unit. Total RNA was extracted using a two-step procedure and samples were analysed using real-time RT-PCR assays for a range of osteo-regulatory cytokines. Results: Human PDL cells expressed mRNA for a range of cytokines of known significance to osteogenesis and osteoclastogenesis in response to mechanical stimulation. Conclusions: The production of osteo-regulatory cytokines by PDL cells in response to mechanical strain suggests that these cells have the potential to contribute to the osseous modeling of orthodontic tooth movement. The presence of osteogenic signalling drive in response to tensile strain tends to support the basic assertions of the pressure-tension hypothesis.

This thesis presents a) a scheme for selecting the most suitable flexure hinge compliance equations, and b) a simple methodology of deriving kinetostatic models of micro-motion stages by incorporating the scheme mentioned above. There were various flexure hinge equations previously derived using different methods to predict the compliances of circular flexure hinges. However, some of the analytical/empirical compliance equations provide better accuracies than others depending on the t/R ratios of circular flexure hinges. Flexure hinge compliance equations derived previously using any particular method may not be accurate for a large range of t/R ratios. There was no proper scheme developed on how to select the most suitable and accurate hinge equation from the previously derived formulations. Therefore, the accuracies and limitations of the previously derived compliance equations of circular flexure hinges were investigated, and a scheme to guide designers for selecting the most suitable hinge equation based on the t/R ratios of circular flexure hinges is presented in this thesis. This thesis also presents the derivation of kinetostatic models of planar micromotion stages. Kinetostatic models allow the fulfillment of both the kinematics and the statics design criteria of micro-motion stages. A precise kinetostatic model of compliant micro-motion stages will benefit researchers in at least the design and optimisation phases where a good estimation of kinematics, workspace or stiffness of micro-motion stages could be realised. The kinetostatic model is also an alternative method to the finite-element approach which uses commercially available software. The modelling and meshing procedures using finite-element software could be time consuming. The kinetostatic model of micro-motion stages wasdeveloped based on the theory of the connection of serial and parallel springs. developed based on the theory of the connection of serial and parallel springs. The derivation of the kinetostatic model is simple and the model is expressed in closed-form equations. Material properties and link parameters are variables in this model. Compliances of flexure hinges are also one of the variables in the model. Therefore the most suitable flexure hinge equation can be selected based on the scheme aforementioned in order to calculate the kinetostatics of micro-motion stages accurately. Planar micro-motion stages with topologies of a four-bar linkage and a 3-RRR (revolute-revolute-revolute) structure were studied in this thesis. These micromotion stages are monolithic compliant mechanisms which consist of circular flexure hinges. Circular flexure hinges are used in most of the micro-motion stages which require high positioning accuracies. This is because circular flexure hinges provide predominantly rotational motions about one axis and they have small parasitic motions about the other axes. The 3-RRR micro-motion stage studied in this thesis has three-degrees-of-freedom (DOF). The 3-RRR stage consists of three RRR linkages and each RRR linkage has three circular flexure hinges. A Pseudo-Rigid-Body-Model (PRBM), a kinetostatic model and a two-dimensional finite-elementanalysis (FEA) model generated using ANSYS of micro-motion stages are presented and the results of these models were compared. Advantages of the kinetostatic model was highlighted through this comparison. Finally, experiments are presented to verify the accuracy of the kinetostatic model of the 3-RRR micromotion stage. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1289361 / Thesis (Ph.D.) -- University of Adelaide, School of Mechanical Engineering, 2007

<p>Wireless sensor networks offer easy implementation, flexibility and mobility of hand held </p><p>devices. Sensors consist of an internal power source, which is the great limitation for </p><p>the life time and the usage of sensor networks. To increase the life time, sensors should </p><p>stay in energy saving sleep mode as long as possible, because in sleep mode the radio is </p><p>either shut down or working with less energy. Better energy handling is implemented in </p><p>different power saving mechanism of common Medium Access Control protocols, which are </p><p>evaluated and analyzed and further extensions and ideas to improve the energy efficiency </p><p>are presented. Slotted PSM is simulated with the NS2 and compared to the WLAN 802.11 </p><p>PSM technology and the results show that energy efficiency and power consumption are </p><p>much better implemented and life time increases with the use of Slotted PSM.</p>

Two novel and highly related C₂H₂ zinc finger proteins (CTIP1/BCL11A/EVI9 and CTIP2/BCL11B/Rit1) have been implicated in COUP-TF signaling, etiology of myeloid and lymphoid malignancies, and hematopoietic cell development. However, the precise cellular function(s) and the contribution of these proteins to neoplastic processes and hematopoietic cell development remain unknown. The goal of the studies described herein was to elucidate the molecular mechanisms underlying the transcriptional repression mediated by these proteins to understand their biological properties, and ultimately, their cellular function(s). CTIP proteins repressed transcription of a reporter gene in a TSA-insensitive manner, suggesting that this repression mechanism(s) may not involve TSA-sensitive histone deacetylation catalyzed by member(s) of class I and II HDACs. One possible mechanism is that CTIP proteins may exert ISA-insensitive histone deacetylation catalyzed by TSA-insensitive HDAC(s), such as SIRT1, to repress transcription. In deed, SIRT1 was found to interact with CTIP proteins both in vitro and in mammalian cells, and was recruited to the promoter template in a CTIP-dependent manner. The proline-rich regions of CTIP proteins and the sirtuin homology domain of SIRT1 were found to be essential for mediating CTIPs•SIRT1 interactions. Moreover, column chromatography revealed that SIRT1 and CTIP2 were components of a large complex in Jurkat cell nuclear extracts. Based on the findings that SIRT1 associates with CTIP proteins in mammalian cells, SIRT1 may underlie the transcriptional repression activity of CTIP proteins. The following results support the hypothesis that SIRT1 may underlie the mechanism(s) of CTIP-mediated transcriptional repression. First, CTIP-mediated transcriptional repression was inhibited, at least partially, by nicotinamide, an inhibitor of the NAD⁺-dependent, TSA-insensitive HDACs. Second, the decrease in levels of acetylated histones H3 and/or H4 at the promoter region of a reporter gene was observed upon overexpression of CTIP proteins, and this effect was inhibited, at least partially, by nicotinamide. Third, endogenous SIRT1 was recruited to the promoter template of a reporter gene in mammalian cells upon overexpression of CTIP proteins. Fourth, SIRT1 enhanced the transcriptional repression mediated by CTIP proteins and this enhancement required the catalytic activity of SIRT1. Finally, SIRT1 enhanced the deacetylation of template-associated histones H3 and/or H4 in CTIP-transfected cells. In summary, results described herein strongly suggest that CTIP-mediated transcriptional repression involves the recruitment of SIRT1 to the template, at which the TSA-insensitive, but nicotinamide-sensitive histone deacetylase catalyzes deacetylation of promoter-associated histones H3 and/or H4. These results contribute additional understanding to the molecular mechanisms underlying transcriptional activity of CTIP proteins, which might be helpful for identification and characterization of the target genes under the control of CTIP proteins in cells of hematopoietic system and/or the central nervous system. / Graduation date: 2004

Rat selenoprotein W (SeW) promoter activity was investigated using different concentrations of cadmium, copper, and zinc. Two fragments (404bp and 1265bp) of the SeW promoter, containing a single metal response element (MRE), were ligated into the multiple cloning site of a pGL3-Basic reporter plasmid. The constructs were transfected into cultured rat C6 (glial) and L8 (myoblast) cells and promoter activity measured by means of luciferase reporter gene fused to the SeW promoter fragments in the reporter plasmid. With post-transfection exposure of these cell lines to these metals, copper and zinc, but not cadmium, significantly increased promoter activity of the unmutated 1265bp (not 404bp) construct (p<0.05) only in the C6 cells. Mutation of the MRE sequence abolished promoter response to metal exposure but did not eliminate promoter activity. The results suggest that SeW expression in glial cells can be increased on exposure to copper and zinc and that this response is dependent on the MRE sequence present in the SeW promoter. To understand transcriptional regulation of the SeW gene, we used in vitro binding assays to identify transcription factors that may be involved in the transcriptional regulation of the SeW gene. Using protein from rat C6 (glial) cell nuclear extracts, oligonucleotides containing putative regulatory elements in the SeW promoter, and antibodies, we were able to show that the specificity protein 1(Sp1) transcription factor binds to the Sp1 consensus sequence in the SeW promoter as well as the MRE. However, the MRE, GRE, AP-1 and LF-A1 did not yield any specific binding. Although, competition analysis showed specific binding at the TFII-1 site, super-shift analysis using anti-TFII-1 antibody did not yield any super-shifted band. Therefore the SeW gene may be a target for Sp1 whose interaction with the SeW promoter may activate or repress the transcription of SeW. / Graduation date: 2003

Thesis (PhD (Public Affairs))--University of Pretoria, 2006. / Abstract in English. Includes bibliographical references. Available on the Internet via the World Wide Web.

Ribonucleases play essential roles in RNA metabolism. In Escherichia coli, the extensive degradation of RNAs that are defective or no longer required by the cell is carried out by one of three processive, 3' to 5' exoribonucleases. Relatively unstructured mRNAs are typically degraded by RNase II or PNPase. In contrast, mRNAs containing extensive secondary structure, and the highly structured rRNA and tRNA molecules, are degraded by PNPase and/or RNase R. However, RNase R differs from other exoribonucleases in that it is able to degrade through these structured RNAs without the aid of a helicase activity. Consequently, its mechanism of action is of great interest. In this dissertation, using a variety of specifically designed substrates, I show that a single-stranded overhang, which must be at least 5 nucleotides in length, is required for tight binding and subsequent degradation of double-stranded RNA by RNase R. Moreover, this overhang must be 3' to the duplex indicating that an RNA substrate must thread into the enzyme with 3' to 5' polarity. Using a series of truncated RNase R proteins, I show that the cold-shock domains and the S1 domain contribute to substrate binding. The cold-shock domains appear to play a role in substrate recruitment, while the S1 domain is required to position substrates for efficient catalysis. Furthermore, the nuclease domain alone is sufficient to bind and degrade structured RNAs. This is a unique property of the nuclease domain of RNase R since this domain in RNase II, a paralogue of RNase R, stalls as it approaches a duplex. RNase R binds RNA more tightly within its nuclease domain than RNase II. Through mutagenesis studies, I identify one amino acid, R572, within the nuclease domain of RNase R that contributes to this tight binding and the ability to degrade double-stranded RNA. Furthermore, I found that degradation of structured RNA is strongly dependent on temperature. Based on these data I propose that tight binding allows RNase R to capitalize on the natural thermal breathing of an RNA duplex to degrade structured RNA.

At the dawn of the Large Hadron Collider era, the Brout-Englert-Higgs mechanism remains the most appealing theoretical explanation of the electroweak symmetry breaking, despite the fact that the associated fundamental scalar boson has escaped any direct detection attempt. In this thesis, we consider a particular extension of the minimal Brout-Englert-Higgs scalar sector implemented in the Standard Model of strong and electroweak interactions. This extension, which is a specific, "twisted", realisation of the generic two-Higgs-doublet model, is motivated by a relative phase in the definition of the phenomenologically successful CP and custodial symmetries. Considering extensively various theoretical, indirect and direct constraints, this model appears as a viable alternative to more conventional scenarios like supersymmetric models, and gives grounds to largely unexplored possibilities of exotic scalar signatures at present and future collider experiments.

In the context of sustainable development, steelmakers and automotive manufacturers decided for some years now to join their efforts to promote the development and use of advanced high strength steels such as the present TRIP steels in order to reduce the fuel consumption and emission of greenhouse gas. These multiphase steels contain some retained austenite, a ductile phase that can transform into hard and brittle martensite during a mechanical solicitation. One the one hand, this transformation improves the mechanical properties during plasticity by bringing about an additional work-hardening. On the other hand, the appearence of a hard and brittle phase can give rise to premature cracking after necking. Knowing the good influence of martensitic transformation on the work-hardening, this Thesis starts with the characterisation of the relationship between transformation rates and testing conditions. It appears that, for each testing condition, there is an optimum austenite stability that leads to a maximum uniform strain. After necking under monotonic loading conditions, the damage mechanisms that takes place in these steels has been characterised. It can be concluded that the TRIP-aided steels that present low or moderate austenite stability behave exactly like Dual-Phase steels, in which martensite replaces retained austenite. However, a very stable retained austenite brings about a significant toughness improvement by providing an additional work-hardening contribution in the necking zone. The mechanical behaviour of these steels has also been characterised under cyclic loading conditions. The results indicate that, for particular loading conditions (i.e. low load levels), the martensitic transformation improves the fatigue properties.