There has been an increase in number of human infections by mycobacteria and opportunistic pathogens of the closely related nocardioform bacteria. Frequent multiple drug resistance in these organisms makes it desirable to identify novel targets for antimicrobial agents. Bacteriophages offer one way to do this as analysis of their DNA reveals great diversity in their genetic makeup, suggesting variety in the way they interfere with host cells. Four novel nocardiophages were therefore isolated from soil and characterized. Libraries of their nucleic acid were constructed and screened for clones inhibitory to a nocardioform of the genus Rhodococcus. Nine clones were characterized, and minimum necessary DNA for inhibitory activity sequenced. Of 18 ORFs predicted on these DNAs, 13 could not be assigned a function. Genes similar to ones in databases apparently interfered with DNA metabolism, protein synthesis, or integrity of plasma membrane. This genetic approach may be an efficient and effective way to discover novel targets for antibiotics.

Part I: Target identification studies are one of the most difficult but rewarding challenges in chemical biology. Part I of this thesis describes target identification studies for 5-nitrofuran containing hits. The 5-nitrofurans used in this study were identified in a phenotypic screen for compounds that induced melanocyte cells death in zebrafish. Chapter 1 provides brief overviews on three related areas of the project: 1) the use of zebrafish as a model organism in drug discovery; 2) phenotypic screening using zebrafish and 3) the strategies used in target identification studies. Chapter 2 describes the synthesis of and SAR studies on two series of 5-nitrofuran containing analogues. The design and preparation of biotinylated chemical probes based on the SAR data is also described. These chemical tools are then used in affinity chromatography studies and genetic validation of a potential target (zebrafish Aldh2) of the 5-nitrofuran compounds is reported. Chapter 3 provides a review of the biological and chemical processes that human ALDHs are known to mediate. In addition, small molecules that modulate ALDH2 activity are reviewed. A detailed study of the interaction between 5-nitrofurans and human ALDH2 including in vitro enzymatic assays is described leading to the conclusion that the 5- nitrofurans under study are substrates of human ALDH2. Further mechanism of action investigations using model reactions are also presented. Chapter 4 introduces the use of 5-nitrofuran containing drugs in the clinic and highlights the reported side-effects. Further investigation of the interaction between ALDH2 and 5- nitrofurans in zebrafish and yeast using ALDH2 inhibitors is described. Based on these results, a combination therapy strategy is proposed. Finally, the trypanocidal activity of the newly synthesised 5-nitrofurans is discussed. Experimental details and future work for Part I are presented in Chapters 5 and 6 respectively. Part II: Human sirtuins are associated with various biological functions and diseases, including cancer and neurodegeneration. Previous work from the Westwood Lab has led to the discovery of the tenovins that act as inhibitors of SIRT1 and SIRT2. Part II of the thesis reports the development of potent fixed ring tenovin analogues with high SIRT2 selectivity. Chapter 7 provides a brief review of the biology of human SIRT2 and the reported SIRT2 inhibitors available to date. This is followed by a short summary of the previous work on the tenovins in the Westwood Lab and the design of the fixed ring tenovin analogues. Chapter 8 describes the synthesis of three series of fixed ring tenovin analogues. SAR data is generated based on in vitro enzymatic assays against both SIRT1 and SIRT2 and the prepared analogues showed relatively high potency and selectivity against SIRT2. Further cell-based deacetylation assay are also discussed. All the experimental details are reported in Chapter 9 and Chapter 10 provides with conclusions and proposed future work.

Previous research has shown that simultaneous auditory identification of the target in a visual search task can lead to more efficient (i. e. ?flatter?) search functions (Spivey et al. , 2001). Experiment 1 replicates the paradigm of Spivey et al. , providing subjects with auditory identification of the search target either before (<em>Consecutive</em> condition) or simultaneously with (<em>Concurrent</em> condition) the onset of the search task. RT x Set Size slopes in the <em>Concurrent</em> condition are approximately 1/2 as steep as those in the <em>Consecutive</em> condition. Experiment 2 employs a distractor ratio manipulation to test the notion that subjects are using the simultaneous auditory target identification to ?parse? the search set by colour, thus reducing the search set by 1/2. The results of Experiment 2 do not support the notion that subjects are parsing the search set by colour. Experiment 3 addresses the same question as Experiment 2, but obtains the desired distractor ratios by holding the amount of relevantly-coloured items constant while letting overall set size vary. Unlike Experiment 2, Experiment 3 supports the interpretation that subjects are using the auditory target identification to parse the search set.

Part I: Various image reconstruction algorithms used for subsurface targets are reviewed. It is shown how some approximate wavefield inversion techniques: Stripmap Synthetic Aperture Radar (SAR), Kirchhoff Migration (KM) and Frequency-Wavenumber (FK) migration are developed from various models for wavefield scattering. The similarities of these techniques are delineated both from a theoretical and practical perspective and it is shown that Stripmap SAR is, computationally, almost identical to FK migration. A plane wave interpretation of both Stripmap SAR and FK migration is used to show why they are so similar. The electromagnetic assumptions made in the image reconstruction algorithms are highlighted. In addition, it is shown that, theoretically, FK and KM are identical. Image reconstruction results for KM, Stripmap SAR and FK are shown for both synthetic and experimental Ground Penetrating Radar (GPR) data. Subjectively the reconstructed images show little difference, but computationally, Stripmap SAR (and therefore, FK migration) are much more efficient. Part II: A preliminary investigation into the use of the Singularity Expansion Method (SEM) for use in identifying landmines is completed using a Finite-Difference Time-Domain code to simulate a simplified GPR system. The Total Least Squares Matrix Pencil Method (TLS-MPM) is used to determine the complex poles from an arbitrary late-time signal. Both dielectric and metallic targets buried in lossless and lossy half-spaces are considered. Complex poles (resonances) of targets change significantly when the objects are buried in an external medium, and perturbation formulae for Perfect Electric Conductor (PEC) and dielectric targets are highlighted and used. These perturbation formulae are developed for homogenous surrounding media, and their utilization for the half-space (layered medium) GPR problem causes inaccuracies in their predictions. The results show that the decay rate (real part) of the complex poles is not suitable for identification in this problem, but that with further research, the resonant frequency (imaginary part) of the complex poles shows promise as an identification feature. / February 2005

Previous research has shown that simultaneous auditory identification of the target in a visual search task can lead to more efficient (i. e. ?flatter?) search functions (Spivey et al. , 2001). Experiment 1 replicates the paradigm of Spivey et al. , providing subjects with auditory identification of the search target either before (<em>Consecutive</em> condition) or simultaneously with (<em>Concurrent</em> condition) the onset of the search task. RT x Set Size slopes in the <em>Concurrent</em> condition are approximately 1/2 as steep as those in the <em>Consecutive</em> condition. Experiment 2 employs a distractor ratio manipulation to test the notion that subjects are using the simultaneous auditory target identification to ?parse? the search set by colour, thus reducing the search set by 1/2. The results of Experiment 2 do not support the notion that subjects are parsing the search set by colour. Experiment 3 addresses the same question as Experiment 2, but obtains the desired distractor ratios by holding the amount of relevantly-coloured items constant while letting overall set size vary. Unlike Experiment 2, Experiment 3 supports the interpretation that subjects are using the auditory target identification to parse the search set.

Part I: Various image reconstruction algorithms used for subsurface targets are reviewed. It is shown how some approximate wavefield inversion techniques: Stripmap Synthetic Aperture Radar (SAR), Kirchhoff Migration (KM) and Frequency-Wavenumber (FK) migration are developed from various models for wavefield scattering. The similarities of these techniques are delineated both from a theoretical and practical perspective and it is shown that Stripmap SAR is, computationally, almost identical to FK migration. A plane wave interpretation of both Stripmap SAR and FK migration is used to show why they are so similar. The electromagnetic assumptions made in the image reconstruction algorithms are highlighted. In addition, it is shown that, theoretically, FK and KM are identical. Image reconstruction results for KM, Stripmap SAR and FK are shown for both synthetic and experimental Ground Penetrating Radar (GPR) data. Subjectively the reconstructed images show little difference, but computationally, Stripmap SAR (and therefore, FK migration) are much more efficient. Part II: A preliminary investigation into the use of the Singularity Expansion Method (SEM) for use in identifying landmines is completed using a Finite-Difference Time-Domain code to simulate a simplified GPR system. The Total Least Squares Matrix Pencil Method (TLS-MPM) is used to determine the complex poles from an arbitrary late-time signal. Both dielectric and metallic targets buried in lossless and lossy half-spaces are considered. Complex poles (resonances) of targets change significantly when the objects are buried in an external medium, and perturbation formulae for Perfect Electric Conductor (PEC) and dielectric targets are highlighted and used. These perturbation formulae are developed for homogenous surrounding media, and their utilization for the half-space (layered medium) GPR problem causes inaccuracies in their predictions. The results show that the decay rate (real part) of the complex poles is not suitable for identification in this problem, but that with further research, the resonant frequency (imaginary part) of the complex poles shows promise as an identification feature.

Part I: Various image reconstruction algorithms used for subsurface targets are reviewed. It is shown how some approximate wavefield inversion techniques: Stripmap Synthetic Aperture Radar (SAR), Kirchhoff Migration (KM) and Frequency-Wavenumber (FK) migration are developed from various models for wavefield scattering. The similarities of these techniques are delineated both from a theoretical and practical perspective and it is shown that Stripmap SAR is, computationally, almost identical to FK migration. A plane wave interpretation of both Stripmap SAR and FK migration is used to show why they are so similar. The electromagnetic assumptions made in the image reconstruction algorithms are highlighted. In addition, it is shown that, theoretically, FK and KM are identical. Image reconstruction results for KM, Stripmap SAR and FK are shown for both synthetic and experimental Ground Penetrating Radar (GPR) data. Subjectively the reconstructed images show little difference, but computationally, Stripmap SAR (and therefore, FK migration) are much more efficient. Part II: A preliminary investigation into the use of the Singularity Expansion Method (SEM) for use in identifying landmines is completed using a Finite-Difference Time-Domain code to simulate a simplified GPR system. The Total Least Squares Matrix Pencil Method (TLS-MPM) is used to determine the complex poles from an arbitrary late-time signal. Both dielectric and metallic targets buried in lossless and lossy half-spaces are considered. Complex poles (resonances) of targets change significantly when the objects are buried in an external medium, and perturbation formulae for Perfect Electric Conductor (PEC) and dielectric targets are highlighted and used. These perturbation formulae are developed for homogenous surrounding media, and their utilization for the half-space (layered medium) GPR problem causes inaccuracies in their predictions. The results show that the decay rate (real part) of the complex poles is not suitable for identification in this problem, but that with further research, the resonant frequency (imaginary part) of the complex poles shows promise as an identification feature.

Murine cytomegalovirus (MCMV) encodes a non-coding RNA, m169, that inhibits the cellular miRNA, miR-27. Previous studies have shown that the overexpression of miR-27 in vitro suppresses replication of MCMV and degradation of miR-27 by m169 is important for the viral replication during the lytic stage of infection in vivo. To understand why the virus specifically targets this cellular miRNA for degradation, this thesis focuses on identification of cellular target genes of miR-27 that are involved in viral growth in the lytic infection. Microarray analysis was conducted to globally examine cellular genes differentially expressed following miR-27 overexpression or repression during MCMV infection. Data obtained from the microarray analysis were analysed in order to select potential targets of miR-27 for functional screening. Functional screening involved siRNA knockdown of individual genes followed by infection with a GFP reporter virus (GFP-MCMV) to assess the effects on viral growth. Knockdown of 5 out of 55 genes (Rpl18a, Lyar, Itga5, Mapkapk3 and Pik3r1) led to a significant reduction in GFP expression. Based on luciferase reporter assays, Mapkapk3 was validated as a direct target of miR-27 with a seed site interaction in its 3’UTR. Mutation of this site in the mRNA was shown to eliminate miR-27-mediated repression. Analysis of MAPKAPK3 protein levels upon infection demonstrates that the protein levels are higher in cells infected with wild type MCMV versus the m169 deletion virus (MCMV Δm169). This is in line with the difference in miR-27 levels in the two infections showing a decrease of miR-27 in wild type MCMV and unaltered levels in MCMV Δm169 infection. Mapkapk3 is a direct downstream target of p38 mitogen-activated protein (MAP) kinase within the p38 MAP kinase pathway, which has previously been shown to be an essential pathway for CMV replication. Expression levels of substrates of MAPKAPK3 including HSP27 and ATF1 were examined during infection to evaluate whether they are regulated by miR-27. The level of phosphorylation of HSP27 was shown to correlate with the levels of MAPKAPK3 during infection and was higher in cells infected with wild type MCMV versus MCMV Δm169. This suggests that MAPKAPK3 and its substrate, HSP27, are regulated by miR-27 during MCMV infection. This work provides an important foundation for further functional studies on the role of Mapkapk3 and its substrates in MCMV infection and its capacity to be dynamically regulated by miR-27. Based on the microarray analysis upon miR-27 overexpression, it was shown that miR-27 has an impact on the cell cycle, consistent with previous studies. Functional analysis of miR-27 in the cell cycle using miR-27 mimics and inhibitors demonstrated that the mimics cause an increase of cells in S phase at early time points (12 and 14 h), whereas the inhibition of miR-27 results in a significant reduction in the S phase population and accumulation of cells in G1 phase. Luciferase reporter assays confirmed that two genes known to be associated with the cell cycle are direct targets of miR-27: polycomb ring finger oncogene 1 (Bmi1) and caveolin 1 (Cav1). Knockdown of Bmi1 and Cav1 leads to a significant decrease in the number of cells in S phase and accumulation of cells in the G1 phase; however, this is the opposite result to that observed with the miR-27 mimics. These results suggest that the increase in cells in the S phase induced by miR-27 mimics is unlikely to be associated with targeting of Bmi1 and Cav1. Furthermore, knockdown of Bmi1 and Cav1 does not affect viral replication in vitro. Since miR-27 induces the transition of cells from the G1 to S phase, further studies are required to identify the miR-27 targets involved in this function. To identify direct targets of miR-27 through biochemical methods, one chapter of this thesis was devoted to developing CLASH datasets (cross-linking, ligation and sequencing of hybrid). This technique can directly map miRNA-mRNA interactions within the Argonaute protein (AGO). Initially, a NIH 3T3 stable cell line expressing AGO2 with a double affinity tag at the N terminus was generated. Analysis of the stable cell line revealed no significant alteration of miR-27 regulation or change in permissiveness to MCMV compared to wild type cells, making this amenable to further studies. Using the stable cell line, the CLASH protocol was carried out and preliminary data collected. In summary, this thesis identifies a direct target of miR-27, Mapkapk3, that is an important gene in MCMV replication that requires further investigation. Mapkapk3 is a substrate of p38 in the p38 MAP kinase pathway which is a signal transduction mediating numerous biological processes in response to cellular stresses including CMV infection. Furthermore, miR-27 overexpression was found to stimulate the G1/S transition of the cell cycle, and miR-27 inhibition had the opposite effect. Previous evidence has shown that MCMV and HCMV arrest the cell cycle in the G1 phase and inhibit host DNA synthesis to create an optimal condition for viral gene expression and DNA replication. Given that MCMV arrests host cells in the G1 phase, it is possible that degradation of miR-27 by MCMV contributes to this effect. Since miR-27 regulates both Mapkapk3 and the cell cycle, it seems likely that a number of targets and pathways underlie the antiviral properties of this miRNA.

No description available.

The need for a non-invasive, accurate, easy-to-use, and cost-effective colorectal cancer (CRC) detection device is apparent in the low survival rates seen in late-stage diagnoses. Once CRC has progressed past stage I, the 5-year survival rate drops significantly, and treatment options become less favourable. The best way to treat CRC is to catch it early. The development of an RNA-cleaving fluorogenic DNAzyme (RFD) holds the potential to remediate this deficiency. A DNAzyme, called RFD-FN1, was identified from a synthetic random-sequence DNA library to selectively bind to an unknown target associated with Fusobacterium nucleatum, which has been found to be overabundant in pre- and cancerous colorectal tissue and stool. Target recognition by the DNAzyme induces the cleavage of a fluorogenic substrate and generates a fluorescent signal to indicate the presence of the bacterium. This thesis outlines the efforts made towards functionalizing the F. nucleatum-responsive probe in stool samples to create a non-invasive screening test. RFD-FN1 is selective towards a heat-stable F. nucleatum protein, but its limit of detection is only 10^7 CFU/mL. Although able to detect spiked concentrations of F. nucleatum cells in processed stool samples, the use of heat, filtering, centrifugation, antibiotics, culturing or serial dilutions are not sufficient to detect the F. nucleatum that is naturally present in the diseased samples. Experiments designed to enrich the target concentration revealed that the target is not produced consistently in any growing condition tested. Size exclusion chromatography and mass spectrometry analysis identified five potential targets that RFD-FN1 may be responding to. Three candidate targets were cloned and purified, but they failed to induce RFD-FN1’s activity. Due to the COVID-19 pandemic, the purification of the final two proteins was not completed. Purifying the two candidate targets and testing their ability to induce RFD-FN1 represents future research efforts. If the target for the DNAzyme is confirmed, a reselection for a more sensitive DNAzyme, that can function in human stool, can be attempted. / Thesis / Master of Health Sciences (MSc)