Discrepancies between metabolic activity and DNA content as tool to assess cell proliferation in cancer research

Cell proliferation is a critical and frequently studied feature of molecular biology in cancer research. Therefore, various assays are available using different strategies to measure cell proliferation. Metabolic assays such as AlamarBlue, WST-1, and MTT, which were originally developed to determine cell toxicity, are being used to assess cell numbers. Additionally, proliferative activity can be determined by quantification of DNA content using fluorophores, such as CyQuant and PicoGreen. Referring to data published in high ranking cancer journals, 945 publications applied these assays over the past 14 years to examine the proliferative behaviour of diverse cell types. Within this study, mainly metabolic assays were used to quantify changes in cell growth yet these assays may not accurately reflect cellular proliferation rates due to a miscorrelation of metabolic activity and cell number. Testing this hypothesis, we compared metabolic activity of different cell types, human cancer cells and primary cells, over a time period of 4 days using AlamarBlue and fluorometric assays CyQuant and PicoGreen to determine their DNA content. Our results show certain discrepancies in terms of over-estimation of cell proliferation with respect to the metabolic assay in comparison to DNA binding fluorophores.

Matching intention with agency : lessons from practice

The research project described in this paper was designed to explore the potential of a wiki to facilitate collaboration and to reduce the isolation of postgraduate students enrolled in a professional doctoral program at a Queensland university. It was also intended to foster a community of practice for reviewing and commenting on one another’s work despite the small number of students and their disparate topics. The students were interviewed and surveyed at the beginning and during the face-to-face sessions of the course and their wikis were examined over the year to monitor, analyse and evaluate the extent to which the agency of the technology (wiki) mediated their development of scholarly skills. The study showed that students paradoxically eschewed use of the structured wiki and formed their own informal networks. This paper will contend that this paradox arose from a mismatch between the agency of technology and its intended purpose.

Investigation of highly regulated promoters for the production of recombinant proteins in plants

Plants have been identified as promising expression systems for the commercial production of recombinant proteins. Plant-based protein production or “biofarming” offers a number of advantages over traditional expression systems in terms of scale of production, the capacity for post-translation processing, providing a product free of contaminants and cost effectiveness. A number of pharmaceutically important and commercially valuable proteins, such as antibodies, biopharmaceuticals and industrial enzymes are currently being produced in plant expression systems. However, several challenges still remain to improve recombinant protein yield with no ill effect on the host plant. The ability for transgenic plants to produce foreign proteins at commercially viable levels can be directly related to the level and cell specificity of the selected promoter driving the transgene. The accumulation of recombinant proteins may be controlled by a tissue-specific, developmentally-regulated or chemically-inducible promoter such that expression of recombinant proteins can be spatially- or temporally- controlled. The strict control of gene expression is particularly useful for proteins that are considered toxic and whose expression is likely to have a detrimental effect on plant growth. To date, the most commonly used promoter in plant biotechnology is the cauliflower mosaic virus (CaMV) 35S promoter which is used to drive strong, constitutive transgene expression in most organs of transgenic plants. Of particular interest to researchers in the Centre for Tropical Crops and Biocommodities at QUT are tissue-specific promoters for the accumulation of foreign proteins in the roots, seeds and fruit of various plant species, including tobacco, banana and sugarcane. Therefore this Masters project aimed to isolate and characterise root- and seed-specific promoters for the control of genes encoding recombinant proteins in plant-based expression systems. Additionally, the effects of matching cognate terminators with their respective gene promoters were assessed. The Arabidopsis root promoters ARSK1 and EIR1 were selected from the literature based on their reported limited root expression profiles. Both promoters were analysed using the PlantCARE database to identify putative motifs or cis-acting elements that may be associated with this activity. A number of motifs were identified in the ARSK1 promoter region including, WUN (wound-inducible), MBS (MYB binding site), Skn-1, and a RY core element (seed-specific) and in the EIR1 promoter region including, Skn-1 (seed-specific), Box-W1 (fungal elicitor), Aux-RR core (auxin response) and ABRE (ABA response). However, no previously reported root-specific cis-acting elements were observed in either promoter region. To confirm root specificity, both promoters, and truncated versions, were fused to the GUS reporter gene and the expression cassette introduced into Arabidopsis via Agrobacterium-mediated transformation. Despite the reported tissue-specific nature of these promoters, both upstream regulatory regions directed constitutive GUS expression in all transgenic plants. Further, similar levels of GUS expression from the ARSK1 promoter were directed by the control CaMV 35S promoter. The truncated version of the EIR1 promoter (1.2 Kb) showed some differences in the level of GUS expression compared to the 2.2 Kb promoter. Therefore, this suggests an enhancer element is contained in the 2.2 Kb upstream region that increases transgene expression. The Arabidopsis seed-specific genes ATS1 and ATS3 were selected from the literature based on their seed-specific expression profiles and gene expression confirmed in this study as seed-specific by RT-PCR analysis. The selected promoter regions were analysed using the PlantCARE database in order to identify any putative cis elements. The seed-specific motifs GCN4 and Skn-1 were identified in both promoter regions that are associated with elevated expression levels in the endosperm. Additionaly, the seed-specific RY element and the ABRE were located in the ATS1 promoter. Both promoters were fused to the GUS reporter gene and used to transform Arabidopsis plants. GUS expression from the putative promoters was consitutive in all transgenic Arabidopsis tissue tested. Importantly, the positive control FAE1 seed-specific promoter also directed constitutive GUS expression throughout transgenic Arabidopsis plants. The constitutive nature seen in all of the promoters used in this study was not anticipated. While variations in promoter activity can be caused by a number of influencing factors, the variation in promoter activity observed here would imply a major contributing factor common to all plant expression cassettes tested. All promoter constructs generated in this study were based on the binary vector pCAMBIA2300. This vector contains the plant selection gene (NPTII) under the transcriptional control of the duplicated CaMV 35S promoter. This CaMV 35S promoter contains two enhancer domains that confer strong, constitutive expression of the selection gene and is located immediately upstream of the promoter-GUS fusion. During the course of this project, Yoo et al. (2005) reported that transgene expression is significantly affected when the expression cassette is located on the same T-DNA as the 35S enhancer. It was concluded, the trans-acting effects of the enhancer activate and control transgene expression causing irregular expression patterns. This phenomenon seems the most plausible reason for the constitutive expression profiles observed with the root- and seed-specific promoters assessed in this study. The expression from some promoters can be influenced by their cognate terminator sequences. Therefore, the Arabidopsis ARSK1, EIR1, ATS1 and ATS3 terminator sequences were isolated and incorporated into expression cassettes containing the GUS reporter gene under the control of their cognate promoters. Again, unrestricted GUS activity was displayed throughout transgenic plants transformed with these reporter gene fusions. As previously discussed constitutive GUS expression was most likely due to the trans-acting effect of the upstream CaMV 35S promoter in the selection cassette located on the same T-DNA. The results obtained in this study make it impossible to assess the influence matching terminators with their cognate promoters have on transgene expression profiles. The obvious future direction of research continuing from this study would be to transform pBIN-based promoter-GUS fusions (ie. constructs containing no CaMV 35S promoter driving the plant selection gene) into Arabidopsis in order to determine the true tissue specificity of these promoters and evaluate the effects of their cognate 3’ terminator sequences. Further, promoter truncations based around the cis-elements identified here may assist in determining whether these motifs are in fact involved in the overall activity of the promoter.

Fraud detection in ERP systems using scenario matching

ERP systems generally implement controls to prevent certain common kinds of fraud. In addition however, there is an imperative need for detection of more sophisticated patterns of fraudulent activity as evidenced by the legal requirement for company audits and the common incidence of fraud. This paper describes the design and implementation of a framework for detecting patterns of fraudulent activity in ERP systems. We include the description of six fraud scenarios and the process of specifying and detecting the occurrence of those scenarios in ERP user log data using the prototype software which we have developed. The test results for detecting these scenarios in log data have been verified and confirm the success of our approach which can be generalized to ERP systems in general.

Human papillomavirus DNA detected in peripheral blood samples from healthy Australian male blood donors

Recent studies have shown that human papillomavirus (HPV) DNA can be found in circulating blood, including peripheral blood mononuclear cells (PBMCs), sera, plasma, and arterial cord blood. In light of these findings, DNA extracted from PBMCs from healthy blood donors were examined in order to determine how common HPV DNA is in blood of healthy individuals. Blood samples were collected from 180 healthy male blood donors (18-76 years old) through the Australian Red Cross Blood Services. Genomic DNA was extracted and specimens were tested for HPV DNA by PCR using a broad range primer pair. Positive samples were HPV-type determined by cloning and sequencing. HPV DNA was found in 8.3% (15/180) of the blood donors. A wide variety of different HPV types were isolated from the PBMCs; belonging to the cutaneous beta and gamma papillomavirus genera and mucosal alpha papillomaviruses. High-risk HPV types that are linked to cancer development were detected in 1.7% (3/180) of the PBMCs. Blood was also collected from a healthy HPV-positive 44-year-old male on four different occasions in order to determine which blood cell fractions harbor HPV. PBMCs treated with trypsin were negative for HPV, while non-trypsinized PBMCs were HPV-positive. This suggests that the HPV in blood is attached to the outside of blood cells via a protein-containing moiety. HPV was also isolated in the B cells, dendritic cells, NK cells, and neutrophils. To conclude, HPV present in PBMCs could represent a reservoir of virus and a potential new route of transmission.

Wide-angle visual feature matching for outdoor localization

Wide-angle images exhibit significant distortion for which existing scale-space detectors such as the scale-invariant feature transform (SIFT) are inappropriate. The required scale-space images for feature detection are correctly obtained through the convolution of the image, mapped to the sphere, with the spherical Gaussian. A new visual key-point detector, based on this principle, is developed and several computational approaches to the convolution are investigated in both the spatial and frequency domain. In particular, a close approximation is developed that has comparable computation time to conventional SIFT but with improved matching performance. Results are presented for monocular wide-angle outdoor image sequences obtained using fisheye and equiangular catadioptric cameras. We evaluate the overall matching performance (recall versus 1-precision) of these methods compared to conventional SIFT. We also demonstrate the use of the technique for variable frame-rate visual odometry and its application to place recognition.

Reliability analysis of transform-based stereo matching techniques, and a new matching constraint

Stereo vision is a method of depth perception, in which depth information is inferred from two (or more) images of a scene, taken from different perspectives. Practical applications for stereo vision include aerial photogrammetry, autonomous vehicle guidance, robotics and industrial automation. The initial motivation behind this work was to produce a stereo vision sensor for mining automation applications. For such applications, the input stereo images would consist of close range scenes of rocks. A fundamental problem faced by matching algorithms is the matching or correspondence problem. This problem involves locating corresponding points or features in two images. For this application, speed, reliability, and the ability to produce a dense depth map are of foremost importance. This work implemented a number of areabased matching algorithms to assess their suitability for this application. Area-based techniques were investigated because of their potential to yield dense depth maps, their amenability to fast hardware implementation, and their suitability to textured scenes such as rocks. In addition, two non-parametric transforms, the rank and census, were also compared. Both the rank and the census transforms were found to result in improved reliability of matching in the presence of radiometric distortion - significant since radiometric distortion is a problem which commonly arises in practice. In addition, they have low computational complexity, making them amenable to fast hardware implementation. Therefore, it was decided that matching algorithms using these transforms would be the subject of the remainder of the thesis. An analytic expression for the process of matching using the rank transform was derived from first principles. This work resulted in a number of important contributions. Firstly, the derivation process resulted in one constraint which must be satisfied for a correct match. This was termed the rank constraint. The theoretical derivation of this constraint is in contrast to the existing matching constraints which have little theoretical basis. Experimental work with actual and contrived stereo pairs has shown that the new constraint is capable of resolving ambiguous matches, thereby improving match reliability. Secondly, a novel matching algorithm incorporating the rank constraint has been proposed. This algorithm was tested using a number of stereo pairs. In all cases, the modified algorithm consistently resulted in an increased proportion of correct matches. Finally, the rank constraint was used to devise a new method for identifying regions of an image where the rank transform, and hence matching, are more susceptible to noise. The rank constraint was also incorporated into a new hybrid matching algorithm, where it was combined a number of other ideas. These included the use of an image pyramid for match prediction, and a method of edge localisation to improve match accuracy in the vicinity of edges. Experimental results obtained from the new algorithm showed that the algorithm is able to remove a large proportion of invalid matches, and improve match accuracy.