The emergence of MNAzymes (Multi-component nucleic acid enzymes) provides a new approach for detection of target analytes in various applications. In this thesis, three novel MNAzyme-based methodologies were developed to expand the range of the applications of MNAzymes. MNAzymes can be coupled with DNA or RNA ligands called aptamers to generate an apta-MNAzyme system, which can be used for the detection of non-nucleic target analytes such as small molecules and proteins. Direct detection using apta-MNAzyme system is performed in a format, which was isothermal, fluorescent, rapid, and requires no protein enzymes. Apta-MNAzymes can be coupled with a signal amplification cascade to increase the sensitivity of the reaction. Another MNAzyme-based methodology termed truncated MNAzyme arm system was developed to discriminate the presence of a single base mismatch of two closely related sequences. The system employs a partzyme with a truncated sensor arm and a stabiliser oligonucleotide that binds adjacently to the truncated sensor arm to stabilise the active MNAzyme structure. Truncated MNAzyme real-time PCR system is capable of discriminating the presence of a single base mismatch in a target DNA with high specificity and sensitivity (down to approximately 10 gene copies). The generic nature of the system enables simultaneous detection of three SNP targets in a multiplex format. MNAzymes was also investigated with various strategies to discriminate DNA sequences that are either methylated or unmethylated. In this thesis, bisulphite-treated DNA samples present in as low as 0.032 % of methylated DNA in a background of unmethylated DNA were discriminated using MNAzyme real-time methylation specific PCR (MSP) system. Furthermore, the presence of 5-methylcytosines in a target sequence increases the melting temperature of the duplex DNA. This was exploited further to directly discriminate DNA methylation status of target sequences using the truncated MNAzyme arm system without the need for bisulphite modification. Findings in this thesis have broadened the scope of MNAzymes as versatile tools for many possible applications and flexible alternative to the current technologies.

Introduction of Doppler ultrasound techniques into the Intensive Care setting has revolutionised the way haemodynamic status is monitored in the critically ill. However, in order to increase the usefulness of these techniques, the Doppler signal and its spectrum need to be further analysed in ways to facilitate a better clinical response. Extensive processing of the Doppler spectrum on Diagnostic ultrasound machines is limited by the real time performance considerations. It was therefore proposed that the spectral information from these systems be extracted off-line and full set of analytical tools be made available to evaluate this information. This was achieved by creating an integrated and modular software tool called Spectron, which was intended as an aid in the overall management of the patients. The modular nature of Spectron was intended to ensure that new analytical tools and techniques could be easily added and tested. The software provides its users with considerable latitude in choosing various data acquisition and analysis parameters to suit various clinical situations and patient requirements. Spectron was developed under the Windows environment to provide a user friendly interface and to address a range of programming problems such as memory management and the size of the colour palettes. Spectron is able to detect the maximal velocities and compute the mean and median velocities. Relative increases in maximal velocities in cardiac blood flows after the administration of inotropic drugs have been shown in the pilot studies that were conducted. Spectron is able to help in obtaining estimates of the aortic blood flows and in other applications such measuring vascular impedance. Stenotic blood flows can be detected by using the spectral broadening index and blood flow characteristics can be studied by using various blood flow indices. Thus, this project attempted to help in patient management by providing clinicians with a range of blood flow parameters and has succeeded in meeting its objective to a large extent / Master of Engineering (Hons)

Wheat is the most economically important crop forming one quarter of Australian farm production. The wheat industry is severely affected by diseases, with fungal pathogens causing the most important economic losses in Australia. The application of fungicides and chemicals can control crop diseases to a certain extent, however, it is expensive and public concern for the environment has led to alternative methods of disease control to be sought, including the use of microorganisms as biological control agents. Microorganisms are abundant in the soil adjacent to plant roots (rhizosphere) and within healthy plant tissue (endophytic) and a proportion possess plant growth promotion and disease resistance properties. Actinobacteria are gram-positive, filamentous bacteria capable of secondary metabolite production such as antibiotics and antifungal compounds. A number of the biologically active endophytes belonging to the Actinobacteria phylum were isolated in our laboratory. A number of these isolates were capable of suppressing the wheat fungal pathogens Rhizoctonia solani, Pythium sp. and Gaeumannomyces graminis var. tritici, both in vitro and in planta indicating the potential for the actinobacteria to be used as biocontrol agents. The aim of this research was to investigate the molecular mechanisms underlying this plant-microbe interaction. The indigenous microbial populations present in the rhizosphere and endophytic environment are critical to plant health and disruptions of these populations are detrimental. The culture-independent technique Terminal Restriction Fragment Length Polymorphism (T-RFLP) was used to characterise the endophytic actinobacteria population of wheat roots under different conditions. Soils which support a higher number of indigenous microorganisms result in wheat roots with higher endophytic actinobacterial diversity and level of colonisation. Sequencing of 16S rRNA gene clones, obtained using the same actinobacteria-biased PCR primers that were used in the T-RFLP analysis, confirmed the presence of the actinobacterial diversity, and identified a number of Mycobacterium and Streptomyces species. It was found that the endophytic actinobacterial population of the wheat plants contained a higher diversity of endophytic actinobacteria than reported previously, and that this diversity varied significantly among different field soils. The endophytic actinobacteria have previously been shown to protect wheat from disease and enhance growth when coated onto the seed before sowing. As the endophytes isolated were recognised as potential biocontrol agents, the impact on the indigenous endophytic microbial population was investigated. Utilising the T-RFLP technique it was established that the use of a commercial microbial inoculant, containing a large number of soil bacterial and fungal strains applied to the soil, disrupts the indigenous endophyte population present in the wheat roots. The hypothesis is that non-indigenous microbes proliferate and dominate in the soil preventing a number of endophytic-competent actinobacterial genera from access to the seed and ultimately endophytic colonisation of the wheat roots. This dramatically reduces diversity of endophytes and level of colonisation. In contrast the use of a single endophytic actinobacteria endophyte inoculant results in a 3-fold increase in colonisation by the added inoculant, but does not significantly affect this indigenous population. Colonisation of healthy plant tissues with fungal endophytes has been shown to improve the competitive fitness with enhanced tolerance to abiotic and biotic stress and improved resistance to pathogens and herbivores. In this study the fungal endophyte population of wheat plants grown in four different soils was analysed using partial sequencing of 18S rRNA gene sequences. Sequence anlaysis of clones revealed a diverse range of fungal endophytes. In this diverse range of fungal endophytes a number sequences were highly similar to those of previously known fungal phytopathogens. A number of sequences detected were similar to fungal species previously identified in soil or plant material but not as endophytes. The remaining sequences were similar to fungal species without a known relationship with plants. Plants have developed an inducible mechanism of defence against pathogens. In addition to local responses plants have developed a mechanism to protect uninfected tissue through a signal that spreads systemically inducing changes in gene expression. In the model plant Arabidopsis thaliana activation of the Systemic Acquired Resistance (SAR) pathway and the Jasmonate (JA)/Ethylene (ET) pathway is characterised by the production of pathogenesis-related (PR) and antimicrobial proteins resulting in systemic pathogen resistance. Endophytic actinobacteria, isolated from healthy wheat roots in our laboratory, have been shown to enhance disease resistance to multiple pathogens in wheat when coated onto the seed before sowing. Real Time RT-PCR was used to determine if key genes in the SAR and JA/ET pathways were induced in response to inoculation with endophytic actinobacteria. Inoculation of wild-type Arabidopsis thaliana with selected strains of endophytic actinobacteria was able to �prime� the defence pathways by inducing low level expression of SAR and JA/ET genes. Upon pathogen infection the defence-genes are strongly up-regulated and the endophyte coated plants had significantly higher expression of these genes compared to un-inoculated plants. Resistance to the bacterial pathogen Erwinia carotovora subsp. carotovora was mediated by the JA/ET pathway whereas the fungal pathogen Fusarium oxysporum triggered primarily the SAR pathway. Further analysis of the endophytic actinobacteria-mediated resistance was performed using the Streptomyces sp. EN27 and Arabidopsis defence-compromised mutants. It was found that resistance to E. carotovora subsp. carotovora mediated by Streptomyces sp. EN27 occurred via a NPR1-independent pathway and required salicylic acid whereas the jasmonic acid and ethylene signalling molecules were not essential. In contrast resistance to F. oxysporum mediated by Streptomyces sp. EN27 occurred via a NPR1-dependent pathway but also required salicylic acid and was JA- and ET-independent. This research demonstrated that inoculating wheat with endophytic actinobacteria does not disrupt the indigenous endophytic population and may be inducing systemic resistance by activating defence pathways which lead to the expression of antimicrobial genes and resistance to a broad range of pathogens.

The activation of 1,25D-dihydroxyvitamin D3 (1,25D) is catalysed by the enzyme 25-hydroxyvitamin D-1ƒhydroxylase (CYP27B1) in the kidney, which is the primary producer of 1,25D in the body. Although the synthesis of 1,25D by CYP27B1 and the catabolism of 1,25D by 25-hydroxyvitamin D-24-hydroxylase (CYP24) also take place in the bone, the significance of the bone cell-specific metabolism of vitamin D remains largely unknown. This thesis investigates the regulation of the expression of CYP27B1, CYP24 and vitamin D receptor (VDR) mRNA, both in the bone and in the kidney, with the aim to determine whether the regulation of the vitamin D metabolism in the bone is independent from that in the kidney. The effects of age, dietary calcium and vitamin D status on the expression these genes in both the kidney and the bone, as well as on a number of biochemical factors known to regulate the renal metabolism of 1,25D, such as PTH, calcium and 1,25D itself, were examined. CYP27B1 mRNA expression was also studied in histological sections of rat femoral bone. Furthermore, CYP27B1, CYP24 and VDR mRNA expression were also identified in specific regions of the rat femur and in a number of bone cell lines, with the aim to identify the bone cell types that have the capacity to metabolise and/or to respond to vitamin D. The age-related decrease in the circulating levels of 1,25D detected in animals ranging in age from 3 weeks to 2 years old, was a direct result of a reduction in the expression of CYP27B1 mRNA and an increase in the expression of CYP24 and VDR mRNA in the kidney. In contrast, the expression of CYP27B1 and CYP24 mRNA in the bone is high from 3 to 15 weeks of age, which is the period of rapid growth and development. The expression of CYP27B1 mRNA in the bone was positively correlated with the circulating levels of calcium throughout aging, which suggests that the 1,25D produced in the bone may be involved in the mineralisation process. The positive correlation found between the expression of CYP27B1 and CYP24 mRNA in the bone was in contrast with the negative correlation found between the expression of these two enzymes in the kidney. This suggests that the 1,25D produced locally in the bone, rather than the 1,25D produced in the kidney, is the primary determinant of the CYP24 activity in the bone. In vitamin D-deplete animals, fed a 0.1% calcium diet (D(-)/LC), the expression of CYP27B1 mRNA was induced and the expression of CYP24 mRNA was suppressed in the kidney. In contrast, both the expression of CYP27B1 and CYP24 mRNA were low in the bones of these D(-)/LC animals. When vitamin D-deplete animals were fed a 1% calcium diet (D(-)/HC), the expression of both CYP27B1 and CYP24 mRNA was high in the bone, which was in direct contrast with the low expression of these genes detected in the kidney. Besides this, a positive correlation was found between the expression of CYP27B1 mRNA in the bone, serum calcium levels and bone mineral volume (BV/TV) in the epiphysis, which supports the findings for the age study that the locally produced 1,25D may be involved in the promotion of bone mineralisation. Although serum PTH levels was positively correlated with the expression of CYP27B1 mRNA in the kidneys of hypocalcaemic animals, there was no such relationship detected between the levels of serum PTH and the expression of CYP27B1 mRNA in the bone. This finding suggests that the regulation of the expression of CYP27B1 mRNA in the bone is different from the regulation found in the kidney. The identification of CYP27B1 mRNA in osteoblasts-like cells, taken together with the associations between serum calcium and CYP27B1 mRNA expression in the previous studies, suggests that 1,25D produced in osteoblasts may play a significant role in the bone mineralisation process. The detection of CYP27B1 mRNA expression in a number of bone marrow cells suggests that locally produced 1,25D may also play a role in the growth and differentiation of hematopoietic cells. / Thesis (Ph.D.)--School of Molecular and Biomedical Science, 2003.

Bibliography : leaves 225-232. / xii, 232 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Computer Science, 1994

Thesis (M.S.)--Ohio University, June, 2007. / Title from PDF t.p. Includes bibliographical references.

<p>Purpose: The aim of this thesis is to study the European (ESTRO/EAU/EORTC) and American (ABS) guidelines how to report the permanent seed implant and the most significant dosimetric parameters. It will also report on the permanent seed implant at Södersjukhuset, Karolinska University Hospital according to the guidelines. A large number of studies on pre- and post-implant dosimetry on permanent seed implants have recently been published but none is considered a standard. This makes it difficult, if not impossible, to compare data from different centres. The differences in reporting will also be discussed in this thesis. Another part of the study is to investigate how the morbidity correlates with the dose. The results in this report will give an overview of the experience at Södersjukhuset.</p><p>Matherials and Methods: This study includes 198 patients who received implants between 2004-2007 with I-125 seeds under transperineal ultrasound at Södersjukhuset (to a prescribed dose of 145 Gy). The dose-planning system VariSeed 7.1 was used with an online connection to the ultrasound system with real-time verification. Dose constraints for the planning system are V(100)>99%, V(150)>60%, V(200)>25%, UrD(10)<130% and UrD(30)<125%. Outer and inner wall of rectum was outlined for 55 patients as recommended by ESTRO/EAU/EORTC and doses to rectum were also computed.</p><p>Results: The median value for dosimetric parameters at Södersjukhuset, Karolinska University Hospital are for the prostate; D(90)=174Gy (153-194Gy), V(100)= 99% (93-100%), V(150)= 57% (40-74%), for the urethra; UrD(30) = 130% (112-147%), UrD(10) = 124% (107-142%) and for the rectum; RD2cc= 98Gy (73-128Gy), RD0.1cc=164Gy (119-240Gy), RV(100)=0.3cc (0.0-1.3cc), RV(150)=0.0cc (0.0-0.2cc). These values correspond to recommended data, except for the V(150) value. Regarding the clinically observed results, 3 patients had a relapse in their cancer, 2 patients had mild proctitis and 15 patients had urinary problems.</p><p>Discussion and Conclusions: The significant dosimetric parameters for reporting according to ESTRO/EAU/EORTC and ABS for prostate are D90[Gy], V(100)[%] and V(150)[%], for urethra are D(30) and D(10), and for rectum RD2cc and RD0.1cc. These parameters consider as a minimum to use and they further recommend secondary parameters to report. Other authors have also recommended to report RV(100) and RV(150) for rectum. This study did not show any relationship between UrD(10), UrD(30) and urinary morbidity. According to the recommendations every patient should undergo a CT-based evaluation. Further investigations are needed on whether a post-implant CT-study is necessary for real-time implantation, as there is not enough published data on this aspect.</p>

<p>This thesis presents new techniques for data management of run-time data objectsin component-based embedded real-time systems. These techniques enabledata to be modeled, analyzed and structured to achieve data managementduring development, maintenance and execution.The evolution of real-time embedded systems has resulted in an increasedsystem complexity beyond what was thought possible just a few years ago.Over the years, new techniques and tools have been developed to manage softwareand communication complexity. However, as this thesis show, currenttechniques and tools for data management are not sufficient. Today, developmentof real-time embedded systems focuses on the function aspects of thesystem, in most cases disregarding data management.The lack of proper design-time data management often results in ineffectivedocumentation routines and poor overall system knowledge. Contemporarytechniques to manage run-time data do not satisfy demands on flexibility,maintainability and extensibility. Based on an industrial case-study that identifiesa number of problems within current data management techniques, bothduring design-time and run-time, it is clear that data management needs to beincorporated as an integral part of the development of the entire system architecture.As a remedy to the identified problems, we propose a design-time data entityapproach, where the importance of data in the system is elevated to beincluded in the entire design phase with proper documentation, properties, dependenciesand analysis methods to increase the overall system knowledge.Furthermore, to efficiently manage data during run-time, we introduce databaseproxies to enable the fusion between two existing techniques; ComponentBased Software Engineering (CBSE) and Real-Time Database ManagementSystems (RTDBMS). A database proxy allows components to be decoupledfrom the underlying data management strategy without violating the componentencapsulation and communication interface.</p> / INCENSE

<p>The usage of Wireless Local Area Networks is increasing rapidly throughout the world. The technology today is not quality proof for the market’s demands. We want to be able to completely wireless perform our demands, such as confer via video or IP-telephony. This is what we call multimedia real-time traffic. It may be achieved over the physical infrastructure in some areas with good results. The goal of this Master’s Thesis is to analyze the possibilities and give solutions and suggestions to achieve multimedia over the wireless networks, with emphasis on the protocol Carrier Sense Multiple Access with Collision Avoidance (CSMA/CA). </p><p>This Master’s Thesis is a theoretical study and the suggested solutions have not been tested in an actual wireless network. Instead they have been tested by computer simulation to give an indication of improvements. Basic configurations are set to the same as in the IEEE 802.11 standard. </p><p>Different methods to reach possible improvements of a WLAN are studied, analyzed and simulated. Such methods are: priority, congestion management and multi-channel protocol. Simulations results show how the priority affects the wireless network and how a multi-channel protocol improves the latency and efficiency of the network. The simulation part is concentrated to show improvements of real-time traffic, which is time sensitive. With a multi- channel protocol the network can allow more users, i.e. more traffic. Also, the network will gain improvement in stability.</p>

<p>Matlab Simulink is a commonly used tool in the design process of control systems. To further take advantage of the Matlab Simulink models it is desirable to translate them for realtime use together with the possibility to read/write physical signals. </p><p>Real-Time Workshop is an extension to Simulink that automatically generates code from a model to a variety of target platforms. RTAI and RTLinux are hard realtime operating systems, making use of Linux. </p><p>To make automatically generated code run on RTAI and RTLinux an adaptation of the generation of code is necessary. </p><p>To control, for example, an automotive engine a data acquisition card with an appropriate device driver is required. Comedi, an open source project, provides a number of device drivers for data acquisition cards. </p><p>The developed system makes use of Simulink, Real-Time Workshop, RTAI or RTLinux, and the standard data acquisition card NI 6035E using a Comedi device driver. The Simulink models may be executed at frequencies up to 50 kHz on ordinary PC hardware. </p><p>The evaluation of the system consisted of measuring the interrupt latency of the used motherboard's bus, measuring computation times running Simulink models with known complexity, running models developed at Vehicular Systems and a comparison of interfacing Simulink/Real-Time Workshop between RTAI and RTLinux. </p><p>The recommended realtime operating system is RTAI due to the open source community support of it as a target platform for Real-Time Workshop.</p>