DEVELOPMENT OF DIGITAL MOLECULAR DIAGNOSTIC ASSAYS FOR MULTIPLEXED NUCLEIC ACID DETECTION AT SINGLE-MOLECULE RESOLUTION

Nucleic acids are the predominant target material in molecular diagnostics, serving as essential biomarkers for genetic diseases, cancers, and pathogen identification. Existing nucleic acid detection methods include nucleic acid amplification tests (NAAT), such as polymerase chain reaction (PCR) and isothermal amplification, and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based approaches. While each method has its distinct advantages and disadvantages, a common limitation they share is the restricted capacity for multiplexing. Despite numerous strategies to enhance the multiplexing capability of current NAAT methods, they often suffer from non-specific exponential amplification and amplification bias, which significantly impact the sensitive detection and accurate quantification of multiple targets. Furthermore, current CRISPR-based genotyping methods require two separate assays with extensive screening of CRISPR guide RNA (crRNA) to precisely determine the mutation status of a specific gene of interest. Digital technology has enhanced the diagnostic capabilities of NAAT as a novel method that distributes each target molecule into microreactors and produces binary signals for absolute quantification of target nucleic acids. Leveraging its single-molecule detection capability, digital technology allows simultaneous detection and quantification of multiple nucleic acid targets with existing multiplexing strategies. Despite significant improvements, existing multiplexed digital assays are often too labor-intensive, rely exclusively on instruments available in limited laboratories, have complicated assay design criteria, or still face the risk of non-specific exponential amplification. There remains a need for simple, reliable, and easily accessible multiplexed nucleic acid detection assays for broad applications. This thesis is devoted to the development of multiplexed molecular diagnostic assays that address the previously mentioned limitations of existing nucleic acid detection methods on multiplexing. We start by outlining various molecular diagnostic methods for nucleic acid detection and discussing their limitations on multiplexing (Chapter 1). Then, we develop a new multiplexing strategy of PCR-based assay that utilizes universal primers and probes with limited fluorescence colors and simple design criteria, termed “Fluorescence-coding PCR” (Chapter 2). Next, we further employ digital technology into the fluorescence-coding PCR for simultaneous detection of multiple nucleic acid targets via single-molecule detection technique (Chapter 3). We also utilize digital technology into the CRISPR-based nucleic acid detection method to expand the potential of CRISPR-based assay on nucleic acid biosensing (Chapter 4), and further develop digitized kinetic analysis method to overcome the ‘one-crRNA-one-target’ limitation and improve the multiplexing capability of CRISPR-based genotyping (Chapter 5)

High Pressure Isotropic Compression and Grain Crushing of Coarse Granular Materials

The effect of grain crushing and grain size on the evolution of water retention curve is investigated based on the framework of Unsaturated Breakage Mechanics (UBM) (Buscarnera &amp; Einav, 2012). The previous study by Gao et al. (2016) have been complemented by additional compression and soil water retention data on two types of granular soils with coarser and finer initial gradings than the ones presented in Gao et al. (2016). The model satisfactorily captures the co-evolution of suction air-entry value (SAEV) and the degree of grain breakage during compression. In addition, this study revealed that the effect of grain shape and pre-yielding void collapse are necessary components for future enhancement of the breakage mechanics model. The compressive and breakage response of coarse granular materials are further investigated using the High Pressure Isotropic Compression (HPIC) device (Mun &amp; McCartney., 2017). This thesis introduces several troubleshooting attempts of the HPIC device and the developed protocols to conduct high-pressure crushing tests. Coarse quartz sand and crushed shale sand are subjected to the high pressure isotropic compression test under dry and saturated states. Differences between the two materials in the compression curve are observed and the possible reasons are discussed. The compression tests are stopped at various stress levels to allow for inspections on the grain size distribution (GSD). Based on these measures, the degree of grain breakage is quantified via Einav&rsquo;s breakage index (Einav, 2007). A clear increase on grain breakage at elevated stress states and the evolution towards an ultimate fractal state is clearly observed. The obtained results are interpreted using a new 1D breakage model to serve as the basis of the next-generation breakage mechanics models.</p

We Should Agree to Disagree

Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.Peer reviewedPublisher PD

Numerical Analysis of Saturated Sand Under Dynamic Loads

In this study, the behavior of reclaimed soils including the effects of excess pore water pressure and the loss of strength under dynamic loads or earthquakes are investigated and simulated. The constitutive model based on the disturbed state concept (DSC) is introduced and DSC-DYN2D program is utilized in a numerical analysis. In the laboratory test, quasi-static and cyclic triaxial tests were carried out to determine parameters for the numerical analysis. Field tests were executed in Inchon International Airport with a 10 tons hydraulic hammer. In the field test, the data of dynamic loads and excess pore water pressure were measured using a 3-D geophone and a pressure transducer respectively, The data of field tests showed the accumulation of excess pore water pressure when rapid dynamic loads were applied. Especially, a back-prediction program based on DSC model is developed and verified its accuracy using various parameters from the cyclic triaxial test. As the results of numerical simulation, the predicted trends for excess pore water pressure compare well with the observed data. Based on the result of this research, it is found that the numerical analysis based on the DSC model is compatible to predict the softening behavior of saturated reclaimed soils under dynamic loads

High speed formation of pyro-carbon coat on silicon carbide fiber by continuous chemical vapor deposition furnace

In order to develop a practical process for the carbon coating on SiC fiber, high-speed and continuous pyrocarbon (PyC) coating process are under development by using a continuous CVD furnace with open-ends structure. The effects of process gas and winding speed have been investigated. It was revealed that the growth rate of PyC is in proportion to winding speed and the flow rate of process gas. PyC growth rate was reached up to 250 nm/min, which is several hundred higher than those of conventional CVI/CVD methods This results indicates that the mass production of PyC coated SiC fiber without unexpected residual deformation (torsion and curvature) is possible. And, the mass production and practical use of SiC/SiC using PyC coated SiC fiber will be largely promoted

Association between body mass index and dental caries in the Kingdom of Saudi Arabia : Systematic Review

Peer reviewe

Cosimulation of MBD (Multi Body Dynamics) and DEM of many spheres using GPU technology

In this paper, dynamic simulation model which have many sphere particles and MBD (Multi Body Dynamics) entities, i.e. bodies, joints, forces, is built and simulated. Many sphere particles are solved with DEM (Discrete Element Method) and simulated with GPU technology. Fast algorithm is applied to calculate hertzian contact forces between many sphere particles (from 100,000 to 1,000,000) and NVIDIA’s CUDA is used to accelerate the calculation. Explicit integration method is applied to solve the many spheres. MBD (Multi Body Dynamics) entities are simulated with recursive formulation. Constraints are reduced by recursive formulation and implicit generalized alpha method is applied to solve dynamic model. Many sphere particles and MBD (Multi Body Dynamics) entities are co-simulated within commercial software RecurDyn. The interaction forces between many sphere particles and rigid body mesh are calculated and applied to each body to simulate two parts simultaneously. These models are built and simulated; fork lifter with sand model, oil in oil tank model, oil filled engine system and water filled washing machine model. All models are simulated with NVIDIA’s GPU and the result is shown

Dental students' knowledge of and attitudes towards prescribing medication in Western Australia

ACKNOWLEDGEMENTThe authors would like to acknowledge UWA dental students for their time and valuable input. Open access publishing facilitated by The University of Western Australia, as part of the Wiley - The University of Western Australia agreement via the Council of Australian University Librarians. [Correction added on 14 May 2022, after first online publication: CAUL funding statement has been added.]Peer reviewedPublisher PD

Australian community pharmacists’ ability to identify the indications for dental prescriptions : A case vignette study

Acknowledgement The authors would like to acknowledge Dr Payne for his assistance in the proofreading of this manuscript. Funding A.T.P. is supported by an NHMRC Early Career Fellowship (1156892)Peer reviewedPublisher PD

Cross-sectional analysis of dental treatment under general anaesthesia in hospitalised Western Australian children in 2018-19

Acknowledgements The authors acknowledge the Department of Dental Medicine staff at the Perth Children’s Hospital. The authors also acknowledge Winthrop 588 Australian Health Review Y. F. A. Alshehri et al. Professor Marc Tennant at the School of Human Sciences and Clinical Associate Professor John Winters from Perth Children’s Hospital for their kind support and assistancePeer reviewedPublisher PD