Metastasis is the leading cause of death in cancer patients. Tumour invasion and migration are critical aspects of metastatic progression. A forward chemical genetics project was initiated in an effort to identify novel compounds that inhibit tumour invasion. After screening a natural extract library, two novel inhibitors were identified: motuporamine C (MotC) and strongylophorine-26 (STP-26). Structure-activity studies identified dihdyromotuporamine C (dhMotC) as a potent and easily synthesized analogue. In this work, the mechanism of activity of dhMotC and STP-26 was investigated. It was found that both dhMotC and STP-26 affect cellular shape. dhMotC induced thick central actin stress fibres and large focal adhesions and caused cells to contract. STP-26 also induced adhesion formation but it reduced stress fibres and caused increased cell spreading. Both inhibitors activated Rho GTPase, a result which was shown to mediate, in large part, the anti-invasion activity of these molecules. Motuporamines also induce the formation of membrane-rich inclusions at the peri-nuclear region in cells. Motuporamines cause an increase in lysosomal pH and inhibit lysosomal function, such that EGFR/EGF complexes internalized in the presence of dhMotC do not get degradad. This inhibition of EGF degradation is not dependent on Rho activity. However, the anti-invasive activity of motuporamine analogues correlates well with their ability to induce the formation of membrane-rich inclusions. Thus, alteration in membrane trafficking or degradation of cellular membranes may be mechanistically related to the anti-invasion effect of motuporamines. A systematic genome-wide yeast haploinsufficiency screen was employed in an effort to identify possible targets of dhMotC. Yeast screening resulted in a list of 21 mutant strains which showed increased drug sensitivity. Sphingolipid biosynthesis was identified as a target in yeast cells. By testing other genes from the list, ARF1 was identified as a target that partially mediates the anti-invasive activity in human cells. The results of this body of work show that Rho and ARF1 are important molecular players in the mechanism of tumour cell invasion. This knowledge will contribute to the development of future anti-metastasis therapies and to the development of small molecules for use as biological probes to investigate the molecular basis of metastasis.

The study of dolphin cognition involves intensive research of animal vocal- izations recorded in the field. In this dissertation I address the automated analysis of audible dolphin communication. I propose a system called the signal imager that automatically discovers patterns in dolphin signals. These patterns are invariant to frequency shifts and time warping transformations. The discovery algorithm is based on feature learning and unsupervised time series segmentation using hidden Markov models. Researchers can inspect the patterns visually and interactively run com- parative statistics between the distribution of dolphin signals in different behavioral contexts. The required statistics for the comparison describe dolphin communication as a combination of the following models: a bag-of-words model, an n-gram model and an algorithm to learn a set of regular expressions. Furthermore, the system can use the patterns to automatically tag dolphin signals with behavior annotations. My results indicate that the signal imager provides meaningful patterns to the marine biologist and that the comparative statistics are aligned with the biologists’ domain knowledge.

Metastasis is the leading cause of death in cancer patients. Tumour invasion and migration are critical aspects of metastatic progression. A forward chemical genetics project was initiated in an effort to identify novel compounds that inhibit tumour invasion. After screening a natural extract library, two novel inhibitors were identified: motuporamine C (MotC) and strongylophorine-26 (STP-26). Structure-activity studies identified dihdyromotuporamine C (dhMotC) as a potent and easily synthesized analogue. In this work, the mechanism of activity of dhMotC and STP-26 was investigated. It was found that both dhMotC and STP-26 affect cellular shape. dhMotC induced thick central actin stress fibres and large focal adhesions and caused cells to contract. STP-26 also induced adhesion formation but it reduced stress fibres and caused increased cell spreading. Both inhibitors activated Rho GTPase, a result which was shown to mediate, in large part, the anti-invasion activity of these molecules. Motuporamines also induce the formation of membrane-rich inclusions at the peri-nuclear region in cells. Motuporamines cause an increase in lysosomal pH and inhibit lysosomal function, such that EGFR/EGF complexes internalized in the presence of dhMotC do not get degradad. This inhibition of EGF degradation is not dependent on Rho activity. However, the anti-invasive activity of motuporamine analogues correlates well with their ability to induce the formation of membrane-rich inclusions. Thus, alteration in membrane trafficking or degradation of cellular membranes may be mechanistically related to the anti-invasion effect of motuporamines. A systematic genome-wide yeast haploinsufficiency screen was employed in an effort to identify possible targets of dhMotC. Yeast screening resulted in a list of 21 mutant strains which showed increased drug sensitivity. Sphingolipid biosynthesis was identified as a target in yeast cells. By testing other genes from the list, ARF1 was identified as a target that partially mediates the anti-invasive activity in human cells. The results of this body of work show that Rho and ARF1 are important molecular players in the mechanism of tumour cell invasion. This knowledge will contribute to the development of future anti-metastasis therapies and to the development of small molecules for use as biological probes to investigate the molecular basis of metastasis.

This thesis describes the development of a small molecule sensor for the detection of specific protein-protein interactions. The responsive sensor has been designed to produce a measurable signal upon a bivalent interaction with a dimeric protein target, and has the potential to probe the biological significance of specific binding events.

This thesis presents a structural model of one aspect of science, the theory-driven discovery of empirical laws, in terms of the knowledge structures and reasoning processes that it involves; and describes a machine learning system designed to embody the major features of the model, called OZ, which is designed to investigate the transport properties of an unknown membrane separating two solutions. Inductive data-driven discovery is an important process in science, but takes place within very tightly constrained limits defined by theoretical reasoning. An explicit specification of the possible search space for a law is a <i>law framework</i>; this takes the form of a law with some undetermined parameters. Inductive law discovery is the search for the values of these free parameters. According to the model <i>informal qualitative models</i> (IQMs) describing the essential structural features of a physical system are used to guide the selection of appropriate variables for scientific law discovery, and the selection of an appropriate mathematical function for a law. Our analysis differs from previous work in machine discovery in stressing the importance of models of internal structure in scientific discovery. OZ comprises a domain independent control structure and a set of domain independent procedures, plus a set of domain dependent heuristics for the membrane properties domain. It constructs a set of candidate IQMs for the unknown membrane, and designs goal-directed experiments to determine which IQM is the right one, generating and testing qualitative predictions about the patterns to be expected in numerical data. When it has identified a single model as correct, it constructs law frameworks for possible laws describing the transport properties of the membrane, then designs different experiments to gather data to supply to an inductive law discovery function, which looks for a law of the type specified by each law framework.

Metastasis is the leading cause of death in cancer patients. Tumour invasion and migration are critical aspects of metastatic progression. A forward chemical genetics project was initiated in an effort to identify novel compounds that inhibit tumour invasion. After screening a natural extract library, two novel inhibitors were identified: motuporamine C (MotC) and strongylophorine-26 (STP-26). Structure-activity studies identified dihdyromotuporamine C (dhMotC) as a potent and easily synthesized analogue. In this work, the mechanism of activity of dhMotC and STP-26 was investigated. It was found that both dhMotC and STP-26 affect cellular shape. dhMotC induced thick central actin stress fibres and large focal adhesions and caused cells to contract. STP-26 also induced adhesion formation but it reduced stress fibres and caused increased cell spreading. Both inhibitors activated Rho GTPase, a result which was shown to mediate, in large part, the anti-invasion activity of these molecules. Motuporamines also induce the formation of membrane-rich inclusions at the peri-nuclear region in cells. Motuporamines cause an increase in lysosomal pH and inhibit lysosomal function, such that EGFR/EGF complexes internalized in the presence of dhMotC do not get degradad. This inhibition of EGF degradation is not dependent on Rho activity. However, the anti-invasive activity of motuporamine analogues correlates well with their ability to induce the formation of membrane-rich inclusions. Thus, alteration in membrane trafficking or degradation of cellular membranes may be mechanistically related to the anti-invasion effect of motuporamines. A systematic genome-wide yeast haploinsufficiency screen was employed in an effort to identify possible targets of dhMotC. Yeast screening resulted in a list of 21 mutant strains which showed increased drug sensitivity. Sphingolipid biosynthesis was identified as a target in yeast cells. By testing other genes from the list, ARF1 was identified as a target that partially mediates the anti-invasive activity in human cells. The results of this body of work show that Rho and ARF1 are important molecular players in the mechanism of tumour cell invasion. This knowledge will contribute to the development of future anti-metastasis therapies and to the development of small molecules for use as biological probes to investigate the molecular basis of metastasis. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Problem: This study is related to the controversy surrounding the relative merits of teaching by discovery and expository methods. Specifically, it investigated the effectiveness of treatment with simple enumeration as a strategy for discovery compared to treatment using an expository method. It was hypothesized that the two treatments would yield the same mathematical achievement, but the simple enumeration treatment would yield more mathematical and non-mathematical transfer effect than the expository treatment. Procedure: The subjects comprised six classes in Mathematics 12. They had been randomly assigned to classes at the beginning of the school year and three classes were assigned to each treatment group. All classes were taught a unit on arithmetic and geometric progressions by the experimenter. Equivalence of the groups was established in terms of the covariates I. Q. and previous term mark. The measuring instruments consisted of the Lorge-Thorndike Intelligence Test, Form 1, Level H of the 1964 Multi-Level Edition; a mathematical content test; and a mathematical transfer test. In addition, the Nonverbal Battery of Form 1 of the Lorge-Thorndike Test was used as a pretest to measure the ability of students to generalize and discover principles from examples. Form 2 was used as a posttest measure to determine whether any improvement in ability to generalize had occurred as a result of the experience with the unit on progressions. The generalized t-test was used to compare means of achievement on all tests. All results were analysed at the University of British Columbia Computing Centre. Conclusions: On the basis of results on the tests, the following conclusions were reached: 1. Treatment with simple enumeration yielded the same level of mathematical achievement as treatment with an expository method. 2. Treatment with simple enumeration yielded significantly greater effect on a mathematical transfer test than treatment with an expository method. An examination of I. Q. levels showed that the superiority in performance was largely located at the medium I. Q. level. 3. Treatment with simple enumeration was no more effective than treatment with an expository method when the criterion measured general transfer. Both groups showed significant improvement in ability to generalize after studying the unit on arithmetic and geometric progressions. The improvement was mainly located at the medium and low I. Q. levels and was independent of teaching method. The implication of this study is that if concern is centred on acquisition of facts, simple enumeration is no more effective than an expository teaching method. However, if there is concern for pupil participation and for training students to advance independently to related but more difficult material, then discovery-orientated lessons are advantageous. / Education, Faculty of / Graduate

Microbial natural products have been an invaluable resource for providing clinically relevant therapeutics for almost a century, including most of the commonly used antibiotics that are still in medical use today. In more recent decades, the need for new biotherapeutics has begun to grow, as multi-drug resistant pathogens continue to emerge, putting into question the long-term efficacy of many drugs that we routinely depend on to combat infectious diseases. To affect this growing medical crisis, new efforts are being applied to computationally mine the genomes of microorganisms for biosynthetic gene clusters that code for molecules possessing anti-microbial activities that circumvent known resistance mechanisms. To this end, cutting-edge software platforms have been developed that can identify, with high predictive accuracy, microbial genomes that code for natural products of potential interest. However, with such analyses comes the need to thoroughly vet each predicted gene cluster, to identify those high-value candidate molecules that are not associated with known resistance mechanisms. In this work, a new strategy was developed that involved cataloguing all known ‘self-resistance’ mechanisms encoded by natural product producing microorganisms, which protect the producer from the highly toxic effects of their secreted anti-microbial agents. This collection of resistance data was leveraged and used to engineer an automated software-based pipeline that interrogates biosynthetic gene clusters and relates them to previously identified resistance mechanisms. Gene clusters that are revealed to be independent of known resistance mechanisms can then be flagged for further chemical and biological study in the laboratory. Such in-depth interrogations of microbial genomes aim to help reveal the full biological repertoire of antibiotics yet to be discovered from microorganisms, and will lead to the development of the next generation of biotherapeutics to quell the growing medical crisis of antibiotic-resistance among human pathogenic organisms. / Thesis / Master of Science (MSc) / It would be hard to imagine a world where we could no longer use the antibiotics we are routinely being prescribed for common bacterial infections. Currently, we are in an era where this thought could become a reality. Although we have been able to discover antibiotics in the past from soil dwelling microbes, this approach to discovery is being constantly challenged. At the same time, the bacteria are getting smarter in their ways to evade antibiotics, in the form of resistance, or self-protection mechanisms. As such is it essential to devise methods which can predict the potential for resistance to the antibiotics we use early in the discovery and isolation process. By using what we have learned in the past about how bacteria protect themselves for antibiotics, we can to stay one step ahead of them as we continue to search for new sources of antibiotics from bacteria.

There is an urgent need to identify novel antibiotics for multidrug-resistant Gram-negative pathogens. These bacteria are intrinsically resistant to many antimicrobials due to a formidable outer membrane barrier. Herein we investigate the potential of perturbing the outer membrane to sensitize Gram-negative bacteria to otherwise inactive antibiotics. In chapter 2, we identify the ability of mcr-1 mediated resistance to confer protection from the lytic but not outer membrane-perturbing activity of colistin. Exploiting this sensitivity, we show that colistin and clarithromycin in combination are efficacious against mcr-1-expressing Klebsiella pneumoniae in murine infection models. This demonstrates the viability of colistin combination therapies against Gram-negative pathogens harbouring mcr-1, and points to a mechanism of mcr-1-mediated resistance extending beyond the predicted reduction in binding affinity of polymyxins to the outer membrane. We continue to investigate the potential of using outer membrane perturbants with otherwise inactive antimicrobials in chapter 3. In this work, we identify the ability of OM disruption to change the rules of Gram-negative entry, render pre-existing resistance ineffective, reduce the development of spontaneous resistance and attenuate biofilm formation. Together, these data suggest that OM disruption overcomes many traditional hurdles encountered during antibiotic treatment and is a high priority approach for further development. / Thesis / Doctor of Philosophy (PhD)

There has been a tremendous rise in the use of Wireless Application Protocol (WAP) services for cellular telephones. Such services include electronic mail, printing, fax delivery, and weather reports. But, current services are limited both in type and nature. Today, mobile telephone users need access to more dynamic, location-based, distributed services that include both hardware resources, like printers and computers, and software services, like application software. Problems due to mobility include clients disconnecting from the network, services leaving the network, and communication problems. This research proposes and demonstrates the feasibility of a framework for a system to meet such a need. More specifically, this work develops and demonstrates a distributed environment where mobile telephone users have access to services dynamically as they enter and leave different service areas. It also provides a framework to support mobility in the application layer context. This work utilizes Sun Microsystem's JINI connection technology to provide distributed services to mobile telephones over WAP. It provides a prototype system to provide Java based software services to mobile telephones. The work also provides several optimizations with respect to client communication by harnessing key features of WAP. This provides a robust, dynamic environment for service provisioning. / Master of Science