title: Biochemical analysis of telomeric repeat binding factor 1

contributor:
Zhu, Xu-Dong
Junop, Murray
Biology

subject:
Telomere
TRF1
phosphorylation
Pin2
Biochemistry
Molecular Biology
Biochemistry

abstract:
TRF1 is an essential shelterin protein that binds to double stranded telomeric DNA. TRF1 is best known for its role as a negative regulator of telomere length. Post-translational modifications, like phosphorylation, have been shown to regulate TRF1 function in cells. Mass spectrometric analysis revealed three potential TRF1 phosphorylation sites, threonine 271, serine 279 and threonine 305 in vivo. To analyze the function of these three potential phosphorylation sites, phosphomimic (aspartic acid, D) and non-phosphorylatable (alanine, A) mutations were made to be analyzed in vitro. Through in vitro gel shift assays, the phosphomimic mutation at threonine 271 mutant exhibits a DNA binding defect, whereas serine 279 and threonine 305 mutants have no binding defects. However, in vivo analysis needs to be conducted in order to determine whether this binding defect is authentic.

PIN2 is a TRF1 isoform, which is identical in its protein sequence with the exception of 20 amino acids (residue 296-316 of TRF1). The second study looks to characterize any potential functional differences between PIN2 and TRF1. In vitro, CDK1 kinase assay was conducted on TRF1 and PIN2 mutants to assess whether there was a difference in phosphorylation. The kinase assay revealed that both PIN2 threonine 351 and TRF1 threonine 371 are both phosphorylated by CDK1. However, the net phosphorylation level of PIN2 by CDK1 is far lower than the net phosphorylation level of TRF1. An in vitro gel shift assay was also conducted to analyze the binding difference between TRF1 threonine 371 and PIN2 threonine 351 mutants. The DNA binding assay revealed that TRF1 threonine 371D mutant has a binding defect, whereas PIN2 threonine 351D mutant has no binding defect. However, this data needs to be verified to determine whether a PIN2 threonine 351D mutant behaves like a phosphomimic.

abstract: Master of Science (MSc)


title: CONTEXTUAL AND INDIVIDUAL LEVEL FACTORS INFLUENCING CHILD HEALTH IN LOW AND MIDDLE INCOME COUNTRIES

contributor: Clinical Epidemiology/Clinical Epidemiology & Biostatistics

abstract:
This thesis examined contextual and individual-level determinants of child health in low and middle income countries, with specific focuses on slum residency, maternal use of cesarean section (CS) during delivery and malaria prevention campaigns. It also examined parental HIV preventive behaviors that could ultimately be associated with child health outcomes (although this was not studied in this thesis). The thesis is presented as four separate projects that examine the following: (1) associations of urban slum residency with infant mortality and child stunting in 45 countries and factors modifying these associations; (2) relations between individual-level CS and neonatal mortality in 46 countries and the moderating influence of country-level CS rates on these relations; (3) determinants of consistent condom use based on the perspectives of both husband and wife where at least one partner is HIV infected and resident in three sub-Saharan African countries with high HIV prevalence; and (4) evaluation of the associations between long-lasting insecticidal nets (LLIN) mass distribution campaigns led by different organizations (the World Bank, UNICEF, or Global Fund) and malaria among children in Nigeria. Data were from nationally representative Demographic and Health Surveys (DHS) for the first three projects and Nigeria Malaria Indicator Survey (NMIS) for the fourth project. The research objectives were addressed using multilevel modeling for projects 1, 3, and 4 and propensity score matching, meta-analysis and meta-regression for project 2.

Results of each separate project of this thesis showed that: (1) after controlling for the socio-economic circumstances of individual families, residency in a slum community was associated with infant mortality. This association was attenuated among children born to women who had received antenatal care from a health professional. Finally, residency in a slum community exacerbated the risk of stunting among older versus younger children; (2) individual-level CS increased the risk of neonatal mortality and that this risk was the highest in countries with the lowest CS rates; (3) couples were more likely to have used condoms consistently if the husband was HIV positive, and was the only one in the couple who knew his HIV test result. This association was stronger if the HIV-positive husband was aware of his HIV test result and his HIV-negative wife aware of her test result. No corresponding associations were observed for HIV-positive wives; (4) compared with children living in areas with no LLIN mass distribution campaigns, those in the World Bank Booster Project areas were significantly less likely to test positive for malaria but no significant differences in child malaria infections were found between other campaigns and non-campaign areas. Results also showed that community-level wealth, community-level maternal knowledge regarding malaria prevention, and child-level use of insecticidal nets were negatively associated with child malaria.

Taken together, the findings suggest that improving the material circumstances of slum neighborhoods and increasing antenatal care coverage among women living in these neighborhoods could help reduce stunted child growth and infant mortality associated with slum residency. Results also indicate the need of improving the quality of maternal and newborn health care services, especially in countries with low CS rates in order to reduce neonatal mortality associated with CS. In addition to LLIN mass distribution campaigns, improving maternal knowledge on malaria prevention at the community level might be helpful in preventing child malaria. Finally, results show that increasing HIV testing and awareness of results among husbands could help reduce HIV transmission among couples.

abstract: Doctor of Philosophy (PhD)


title: Vitamin D and Respiratory Tract Infections (RTIs): The Impact of Vitamin D on the Risk and Severity of Upper RTIs and the Role of Vitamin D in Influenza Vaccine Immunogenicity in Children

contributor:
Loeb, Mark
Walter, Stephen
Smieja, Marek
Health Research Methodology

subject:
Vitamin D
Serum 25-hydroxyvitamin D
Respiratory tract infection
influenza vaccine
Hutterite communities
Infectious Disease
Infectious Disease

abstract:
Recent evidence suggests that vitamin D may be important for immune function. Canadian studies have reported varying prevalences of low levels of vitamin D. Whether these low vitamin D levels are associated with susceptibility to respiratory tract infections (RTIs) and infection severity remains unclear given the inconsistent association in recent studies. Influenza virus as a cause of RTI is of particular interest given its prevalence, morbidity and economic burden. Vaccination is a key strategy in prevention, but little is known about the effect of vitamin D on influenza vaccine response.

A prospective cohort study of children 3 to 15 years old living in Hutterite communities in Alberta, Saskatchewan and Manitoba was conducted to assess the prevalence and predictors of low vitamin D levels and evaluate the association between vitamin D and the incidence and severity of laboratory proven respiratory tract infections. In those who received influenza vaccination, the relationship between vitamin D and influenza vaccine immunogenicity was examined.

A total of 743 children were included in the study. The median serum 25-hydroxyvitamin D level (25[OH]D) was 62.0 nmol/L (interquartile range 51.0, 74.0). Levels lower than 50 nmol/L were present in 152 children (20.5%) and lower than 75 nmol/L in 565 children (76%). Lower serum 25(OH)D levels were associated with increased risk of RTI. No association was found between serum 25(OH)D level and disease severity. There was also no relationship found between serum 25(OH)D level and seroprotection or seroconversion from inactivated influenza vaccine.

In conclusion, low serum 25(OH)D levels are a significant problem in Canadian Hutterite communities. Furthermore, low serum 25(OH)D levels were associated with increased risk of proven upper RTIs. Studies evaluating the role of vitamin D supplementation to reduce the burden of disease are warranted, and strategies to improve vitamin D status in rural communities in Canada are needed.

abstract: Master of Science (MSc)


title: Role of RanBPM in Drosophila fat body for the remote control of insulin secretion and feeding behavior

contributor:
Campos, Ana R.
Roger Jacobs and Andre Bedard
Biology

subject:
Biology
Developmental Biology
Genetics
Biology

abstract:
Abstract

RanBPM (Ran-Binding Protein in the Microtubule Organizing Center) is an evolutionarily conserved protein presumed to function as a scaffold in various signal transduction pathways. Null mutations in the Drosophila RanBPM gene cause a pleiotropic effect as seen by the late larval lethality, impaired growth, reduced CNS proliferation and disruption in feeding and response to light behaviours (Scantelbury et al., 2010). We report that RanBPM mutants display impaired expression and secretion of one of the major Drosophila insulin like peptides, DILP2. Consistent with these findings RanBPM mutants showed reduced membrane localization of the Phosphatidylinositol (3,4,5)-trisphosphate (PIP3) reporter TGPH (GFP-pleckstrin homology domain (PH) fusion regulated by the Tubulin promoter) and reduced activation of the serine threonine kinase AKT, both read outs of insulin signalling (InS). Ubiquitous overexpression of DILP2 partially rescued the reduced larval growth, CNS proliferation and feeding phenotypes caused by RanBPM mutations. Over expression of DILP6 in the glia rescued the reduced CNS proliferation but not the reduced larval growth and feeding behaviour phenotypes. Targeted expression of RanBPM in the fat body suppressed all RanBPM mutant phenotypes except for lethality. In Drosophila, the fat body acts a nutritional sensor involved in coordinating neuroblast proliferation by a TOR- dependent mechanism. Our results present evidence for the role of AKT in the fat body of the RanBPM mutants for the regulation of CNS proliferation, DILP2 secretion, overall growth and feeding

abstract: Master of Science (MSc)


title: Development of neurotransmission in the lateral superior olive: understanding synapse maturation in the developing auditory brainstem

contributor:
Gillespie, Deda
Neuroscience

subject:
circuit development
synaptic development
auditory system
neurophysiology
Developmental Neuroscience
Systems Neuroscience
Developmental Neuroscience

abstract:
The lateral superior olive (LSO) is an auditory brainstem nucleus crucial in the determination of sound source. To accomplish sound localization, principal neurons of the LSO compare the intensity of sounds reaching the two ears by integrating an excitatory input from the ipsilateral anteroventral cochlear nucleus (AVCN), which is activated by sound reaching one ear, with an inhibitory input from the ipsilateral medial nucleus of the trapezoid body (MNTB), which is activated by sound reaching the opposite ear. In order for LSO principal neurons to properly integrate these excitatory and inhibitory inputs, the inputs must be matched in a frequency-dependent matter to LSO neurons. The mechanisms that direct the organization, selection, and maturation of both the excitatory and inhibitory pathway during development are not well understood. The experiments presented in this thesis were aimed at understanding the mechanisms that may underlie these processes in the developing LSO.

The excitatory neurotransmitter glutamate is released in both the excitatory AVCN-LSO pathway and the inhibitory MNTB-LSO pathway during their period of functional circuit refinement, and may be important in the development of both of these pathways. Using the patch-clamp technique in acute brainstem slices of rats, we evaluated glutamatergic transmission in both the excitatory AVCN-LSO pathway and the inhibitory MNTB-LSO pathway during their period of functional refinement. Additionally, using the patch-clamp technique in acute brainstem slices of mice, we examined what functions vesicular glutamate transporter 3 (VGlut3), the protein that supports glutamate release from MNTB terminals, may have in the developing MNTB-LSO pathway. When taken together, the results from the three studies presented support a model in which circuit maturation in the LSO relies on mechanisms driven through a specific glutamate receptor, the N-methyl-D-aspartate (NMDA) receptor.

abstract: Doctor of Philosophy (PhD)


title: ROLE OF TRANSFORMING GROWTH FACTOR BETA IN PROTEINURIA

contributor: Medical Sciences (Molecular Virology and Immunology Program)

subject:
Medical Molecular Biology
Medical Molecular Biology

abstract:
The incidence and prevalence of people suffering from end stage renal disease is increasing. Proteinuria, particularly the presence of albumin in urine is concerning because proteinuria is associated with the progression to end stage renal disease (ESRD). Understanding the mechanisms involved in damaging the glomerular filtration barrier is essential. Transforming growth factor beta (TGFB) is a key cytokine in mediating glomerulosclerosis and proteinuria. Not much is known about the downstream pathways that mediates the renal damage and proteinuria.

I hypothesize that TGFB induces proteinuria through podocyte de-differentiation and this occurs through SMAD dependent and independent pathways.

Methods: I used adenovirus mediated gene transfer of TGFB1 to rat renal artery to study the effects of TGFB1 on renal structure and functions. To study the importance of SMAD3 in mediating downstream effects of TGFB1 in proteinuria and podocyte effacement, I used an anti-glomerular basement membrane model in SMAD3+/+ and SMAD3-/- mice to induce glomerulonephritis and proteinuria.

Results: Transient TGFB1 overexpression via AdTGFB1 induced significant proteinuria, podocyte foot process effacement, nephrin down-regulation, and nephrinuria. The expression of synaptopodin was also significantly down-regulated by TGFB1. TGFB1 increased the expression of the angiopoietin receptor, Tie2, in podocyte cell culture. In cultured podocytes, TGFB1 downregulated the gene and protein expression of both nephrin and synaptopodin. These findings suggest that locally produced TGFB1 can cause podocyte de-differentiation marked by a loss of synaptopodin, nephrin, and foot process effacement; this process is partly regulated by angiopoietins. This process represents a novel pathway that may explain proteinuria in a variety of common renal diseases.

Both SMAD3+/+ and SMAD3-/- mice had proteinuria after induction of anti-GBM glomerulonephritis, though to a lesser extent in SMAD3-/- mice. SMAD3-/- and SMAD3+/+ mice developed significant glomerulonephritis with progressive interstitial fibrosis and chronic renal impairment. The SMAD3+/+ mice were found to be more prone to fibrotic changes, interstitial damage and tubular and glomerulosclerosis than the SMAD3-/- mice. This suggests that TGFB1 signals through pathways other than SMAD3 such as those triggered by hypoxia.

Conclusion: I have shown that TGFB1 upregulation via AdTGFB1 induces proteinuria through podocyte dedifferentiation and FP effacement. Angiopoietins are essential for TGFB1 mediated podocyte injury. The effects of TGFB are partially mediated through SMAD3 as there is residual podocyte effacement and proteinuria in the SMAD3-/- mice. Hence there are SMAD3 dependent and independent pathways involved in proteinuria.

abstract: Doctor of Science  (PhD)


title: Lithium-Ion Batteries: Modelling and State of Charge Estimation

contributor:
Habibi, Saeid
Ali Emadi, Gillian R. Goward
Mechanical Engineering

subject:
Lithium-Ion
Batteries
Modelling
State of Charge
Estimation
SOC
Computer Engineering
Engineering
Mechanical Engineering
Computer Engineering

abstract:
Lithium-ion (Li-ion) cells are increasingly used in many applications affecting our

daily life, such as laptops computers, cell phones, digital cameras, and other portable

electronic devices. Lithium-ion batteries are increasingly being considered for their use in Electric Vehicles (EV), Hybrid Electric Vehicles (HEV) and Plug-in Hybrid Electric Vehicles (PHEV) due to their high energy density, slow loss of charge when not in use, and for lack of hysteresis effect. New application domains for these batteries has placed greater emphasis on their energy management, monitoring and control strategies.

In this thesis, a comparative study between different models and state of charge (SOC) estimation strategies is performed. Battery models range from black-box representation to detailed electrochemical reaction models that consider the underlying physics. The state of charge is estimated using the Extended Kalman filter (EKF) and the Smooth Variable Structure Filter (SVSF). The models and SOC estimation strategies are applied to experimental results from BMW Electrical and Hybrid Research and Development center and validated using a simulation model from AVL CRUISE software.

Overall, different models and SOC estimation scenarios were studied. An average improvement of 30% in the estimation accuracy was shown by the SVSF SOC method when compared with the EKF SOC strategy. In general, the SVSF SOC estimation technique demonstrates excellent capability and a fast speed of convergence.

abstract: Master of Applied Science (MASc)


title: A Pneumatic Sensor for Grinding Wheel Condition Monitoring

contributor:
Koshy, Philip
Mechanical Engineering

subject:
wheel wear
dressing
vitrified
diamond abrasive
surface condition
Manufacturing
Manufacturing

abstract:
Changes to the surface condition of a grinding wheel caused by excessive wear can result in geometric inaccuracy and severe thermal damage to a workpiece. As a precision metal removal process, grinding is typically a finishing operation and such errors are costly in both materials and lost time. Unfortunately grinding wheel performance is difficult to predict, and workpiece damage is commonly prevented by frequent dressing of the wheel surface. However, such over dressing is also costly in unnecessary machine down time and consumption of the grinding wheel. Monitoring systems have been developed in an effort to prevent damage to the workpiece and unnecessary dressing, but various difficulties have prevented any single system from achieving widespread application.

The following body of research focuses on the investigation and development of a pneumatic sensor for monitoring the surface condition of a grinding wheel. These sensors are relatively simple, robust and inexpensive, and well suited to in situ applications. While these sensors are traditionally used to measure displacement of static or quasi-static surfaces, research into dynamic applications has shown they can detect the features or topography of a moving surface. Inspired by these developments, a monitoring method employing both the static and dynamic measurement capabilities of the sensor is proposed with applications to both metal-bonded and vitrified grinding wheels.

abstract: Master of Applied Science (MASc)


title: A PRACTICAL SIMULATION METHODOLOGY TO IMPROVE FATIGUE LIFE PREDICTION OF ENGINE OIL COOLER UNDERGOING PRESSURE CYCLE TESTING

contributor:
Jain, Mukesh K.
Mechanical Engineering

subject:
engine oil cooler
pressure cycle testing
fatigue life prediction
Computer-Aided Engineering and Design
Computer-Aided Engineering and Design

abstract:
Computer simulation is widely used to predict the fatigue life of engine oil coolers that fail under pressure cycles. The objective of this study is to develop a practical simulation methodology to accurately predict the fatigue life of an engine oil cooler undergoing pressure cycle testing. The study focuses on two key areas of the simulation process. First, it investigates the effect of using linear and nonlinear FEA to provide stress or strain results for subsequent fatigue analysis. Second, due to lack of fatigue material properties for the aluminum coreplate material, approximate material models derived from tensile properties are used in fatigue life calculation. The study has attempted to find out the material model that gives the best correlation in life prediction. The life prediction correlation based on the Seeger, the Modified Universal Slopes and the Modified Mitchell models, together with the Modified Universal Slopes-Al model, are evaluated.

It is concluded that the Modified Universal Slopes-Al model, which is a re-assessment of the Modified Universal Slopes model based on the fatigue data of 16 wrought aluminum alloys, gives the best life prediction for simulations using either linear or nonlinear approaches. Life prediction using nonlinear finite element results together with this approximate material model is recommended to be the best approach. On the other hand, a simple and quick linear analysis, followed by fatigue life calculation using this material model still gives life estimates with an acceptable level of confidence.

In the last part of the study, the life prediction performance using different strain-life criteria, together with either Morrow or Smith-Watson-Topper (SWT) mean stress correction, are evaluated. It is found that SWT mean stress correction method is worse than that of Morrow in EOC fatigue life prediction in both linear and nonlinear approaches. Using the principal strain criterion with SWT mean stress correction gives conservative life prediction in both approaches. On the other hand, there are no significant differences in life prediction correlations using the principal strain, the Brown-Miller combined strain and the maximum shear strain strain-life criteria, with Morrow mean stress correction. As such, the Brown-Miller combined strain criterion with Morrow mean stress correction is the recommended strain-life model used in fatigue life calculation.

abstract: Master of Applied Science (MASc)


title: A Novel Approach to Detecting Listeria monocytogenes: Creating Species-Specific Ribonuclease (RNase)-Cleaved Fluorescent Substrate (RFS) by In Vitro Selection

contributor:
Li, Yingfu
Guarné, Alba
Burrows, Lori
Biochemistry and Biomedical Sciences

subject:
Ribonuclease
SELEX
Biosensor
Enzyme Kinetics
Listeria monocytogenes
Functional nucleic acid
Biochemistry
Biology
Biophysics
Molecular Biology
Biochemistry

abstract: The food-borne pathogen, Listeria monocytogenes, is a global health concern as it has been responsible for multiple food contamination outbreaks over the past century. Current detection methods like the enzyme-linked immunoassays (ELISA), and polymerase chain reaction (PCR) take over 24 h to attain results, are costly, require specialized equipment and trained personnel. In this study we investigated the use of functional nucleic acid (FNA) to develop a rapid and cost-effective detection method for L. monocytogenes. We carried out in vitro selection in order to isolate a fluorescently labeled DNA-RNA hybrid strand that can be bound and cleaved by specific endoribonucleases (RNase) from L. monocytogenes. We termed these DNA-RNA hybrid strands RNase-cleaved fluorescent substrate (RFS). Since no past studies have isolated RNases from L. monocytogenes, we first identified the genes based on sequence similarities with well characterized RNases. We purified and characterized RNase HII, RNase III and RNase G. Since this study focused primarily on developing RFS for RNase HII, we performed an in depth in vitro biochemical analysis to characterize this enzyme. We found that RNase HII from L. monocytogenes plays an important role in DNA replication and repair. Furthermore, we obtained six sequence classes by in vitro selection which could interact with RNase HII. The key nucleotide regions involved with RNase HII interactions were identified. In the final study, we showed the sequences isolated by in vitro selection could also be used as a tool to study ribonuclease function and identify new interaction between enzyme and substrate.

abstract: Master of Science (MSc)


title: EVENT-RELATED POTENTIAL (ERP) RESPONSES TO MUSIC AS A MEASURE OF EMOTION

contributor: Laurel Trainor, Geoffrey Hall,  Norman Buckley

subject:
ERP
music
alexithymia
depression
P300
brain injury
Cognitive Neuroscience
Cognitive Neuroscience

abstract: This thesis examines how ERP responses to music provide an index of emotion in control, alexithymic, depressed and depressed alexithymic individuals using a musical affective priming task, with the objective being its clinical application to assess emotion in brain injured patients. Participants listened to pairs of music primes and music targets (music-music paradigm) and word targets (music-word paradigm) to mentally decide if they matched or not according to emotional valence (happy, sad). Responses manifested in the N300 (emotional categorization), P300 (emotional recognition) and N400 (emotional meaning) ERP components, with larger and more differentiated responses for the music-word paradigm indicating a less automatic nature than the music-music paradigm. Alexithymic individuals showed disrupted responses for all components for sad word targets, indicating a sequence of disconnects producing their decreased awareness of and difficulty with regulating emotion. Depressed participants displayed an emotional negativity bias for sad word targets in the P300 and N400, attributable to difficulty disengaging (cognitive rumination), reflecting how emotional deficits affect awareness. Disrupted P300 and N400 responses in the depressed alexithymic individuals were isolated to alexithymia rather than the emotional negativity bias. Specific processing deficits of happy music targets found only in depressed alexithymic individuals demonstrate how alexithymia increases the severity of depression. Although depression effects are more pervasive, alexithymia modifies depressive effects in emotional regulation. The P300 was most reliably seen in depression, and alexithymia to a lesser extent. Therefore, ERP responses to music can effectively and covertly measure emotion and different levels of automaticity, alexithymia and depression. This thesis is the first to demonstrate: 1) how music conveys emotion in a pure musical context (music-music); 2) musical emotion perception in alexithymia, depression and depressed alexithymia; 3) an effective non-verbal measure of emotion for assessing emotional states in brain injured populations.

abstract: Doctor of Philosophy (PhD)


title: Interpreting the Genetic Revolution: A History of Genetic Counseling in the United States, 1930-2000.

contributor:
Balcom, Karen
History

subject: genetic counseling

subject: geneticization

subject: system of professions

subject: history of health and medicine

subject: social history

subject: oral history

subject: History of Science, Technology, and Medicine

abstract: This dissertation explores the social history of genetic counseling in the United States between 1930 and 2000. I situate genetic counselors at the interstices of medicine, science, and an increasingly “geneticized” society. My study emphasizes two central themes. First, genetic counselors have played a crucial role in bridging the “old eugenics” and the “new genetics” as mediators of genetic reproductive technologies. Genetic counselors negotiated the rights and responsibilities of genetic citizens in their patient encounters. Discourses of privilege and duty were also extrapolated outward to public debates about the new genetics, demonstrating the highly-politicized contexts in which counselors practice and women make reproductive choices. Second, I interrogate the professionalization process of genetic counseling from a field led by male physician-geneticists in the 1940s and 50s, to a profession dominated by women with Masters degrees by the 1980s and 90s. This transformation is best understood through the framework of a “system of professions,” and counselors’ professional position between “sympathy and science.” These frameworks similarly structured the client-counselor relationship, which also centered on concepts of risk, the promotion of patient autonomy, and the ethics of non-directiveness and client-centeredness. These principles distanced counselors from their field’s eugenic origins and the traditional doctor-patient relationship. I emphasize the voices of genetic counselors based on 25 oral history interviews, and hierarchies of gender, race, and educational status at work in the profession’s history. A study of genetic counseling is an important contribution to the histories of health and medicine, medical sociology, bioethics, disability studies, and gender and women’s studies.

abstract: Doctor of Philosophy (PhD)


title: Functional Studies of the Interstrand Cross-link Repair Protein, Pso2

contributor:
Junop, Murray
Biochemistry and Biomedical Sciences

subject:
DNA repair
Interstrand cross-link (ICL)
Pso2
Exonuclease
Endonuclease
Hairpin
Biochemistry
Biochemistry

abstract: DNA interstrand cross-links (ICLs) constitute one of the most severe types of DNA damage. ICLs covalently tether both strands of duplex DNA, preventing unwinding and polymerase access during replication and transcription. This obstruction is exploited in cancer chemotherapy since it leads to replication fork collapse, double strand breaks (DSBs), and cell death. Mechanistic understanding of how eukaryotic cells repair these specific lesions, however, is still in its infancy. It is understood that ICL repair consists of a multitude of intersecting and connecting repair pathways that rely on interplay between critical protein factors. Interestingly, Pso2 has been identified as an integral component of the ICL repair pathway in Saccharomyces cerevisiae. Pso2 is a yeast nuclease from the β-CASP family of proteins that function predominantly in the repair of ICLs. It has been recognized as the only protein that does not serve a redundant function in any other DNA repair pathway. It remains unclear how the ICL repair pathway generates DNA intermediates suitable for high fidelity repair dependent on Pso2 nuclease activity. Here we show that Pso2 possesses structure-specific endonuclease activity that may be essential to its role in ICL repair. Direct in vitro activity assessment of the protein on a site-specific ICL proved to be inconclusive due to the heat-labile nature of the cross-linking agent employed. In vitro activity testing was also performed on various substrates resembling intermediates generated during ICL repair. Biochemical analysis demonstrated that Pso2 cleaves hairpins, stem loops, heterologous loops, and symmetrical bubbles. Although the precise cleavage sites vary between substrates, Pso2 demonstrates preference for the single- to double-stranded junction in the DNA backbone, with similar activity to that previously demonstrated for its human homologue, Artemis. This specific endonuclease activity is stimulated by increased concentrations of phosphate. Through two-dimensional gel electrophoresis, the presence of unique DNA intermediates generated in response to ICL damage in vivo was also monitored. Results suggest the generation of hairpin-like intermediates that resemble those tested in vitro. These intermediates persist in the absence of Pso2 but are resolved by exogenous addition of control endonucleases. Our findings expand on previous data that established hairpin-opening activity for this protein and suggest that the structure-specific endonuclease activity demonstrated by Pso2 is important for ICL repair. We anticipate that Pso2 acts on a hairpin-containing DNA substrate in the ICL repair pathway and the resolution of this intermediate is uniquely dependent on Pso2 for the effective repair of ICL damage in yeast. Taking into consideration the current models of ICL repair, both in yeast and humans, possible roles for Pso2 have been described. Achieving a complete mechanistic perspective of this pathway is critical for the therapeutic exploitation of the human homologue, SNM1A. Implications include the potential inhibitory target for increased efficacy of chemotherapy with cross-linking agents.

abstract: Master of Science (MSc)


title: A statistical framework to detect gene-environment interactions influencing complex traits

contributor:
Paré, Guillaume
Canty, Angelo
Meyre, David
Health Research Methodology

subject:
genetic epidemiology
gene-environment interactions
variance heterogeneity
genetics
Applied Statistics
Bioinformatics
Biostatistics
Computational Biology
Genetics
Genomics
Statistical Methodology
Applied Statistics

abstract: Advancements in human genomic technology have helped to improve our understanding of how genetic variation plays a central role in the mechanism of disease susceptibility. However, the very high dimensional nature of the data generated from large-scale genetic association studies has limited our ability to thoroughly examine genetic interactions. A prioritization scheme – Variance Prioritization (VP) – has been developed to select genetic variants based on differences in the quantitative trait variance between the possible genotypes using Levene’s test (Pare et al., 2010). Genetic variants with Levene’s test p-values lower than a pre-determined level of significance are selected to test for interactions using linear regression models. Under a variety of scenarios, VP has increased power to detect interactions over an exhaustive search as a result of reduced search space. Nevertheless, the use of Levene’s test does not take into account that the variance will either monotonically increase or decrease with the number of minor alleles when interactions are present. To address this issue, I propose a maximum likelihood approach to test for trends in variance between the genotypes, and derive a closed-form representation of the likelihood ratio test (LRT) statistic. Using simulations, I examine the performance of LRT in assessing the inequality of quantitative traits variance stratified by genotypes, and subsequently in identifying potentially interacting genetic variants. LRT is also used in an empirical dataset of 2,161 individuals to prioritize genetic variants for gene-environment interactions. The interaction p-values of the prioritized genetic variants are consistently lower than expected by chance compared to the non-prioritized, suggesting improved statistical power to detect interactions in the set of prioritized genetic variants. This new statistical test is expected to complement the existing VP framework and accelerate the process of genetic interaction discovery in future genome-wide studies and meta-analyses.

abstract: Master of Health Sciences (MSc)


title: Influence of intestinal microbiota on the postnatal development of enterochromaffin cells and the enteric nervous system

contributor:
Ratcliffe, Elyanne
Murphy, Kathryn
Jane Foster,  Waliul Khan
Neuroscience

subject:
microbiota
enteric nervous system
enterochromaffin cells
dopamine
serotonin
ENS
Digestive, Oral, and Skin Physiology
Digestive, Oral, and Skin Physiology

abstract:
At birth the gastrointestinal (GI) tract is rapidly colonized by microbial organisms which exhibit considerable fluctuations in composition across the first two years of life. During this period, the enteric nervous system (ENS) continues to undergo significant structural and functional changes. In the present study, we investigated whether exposure to intestinal microbiota influences the postnatal development of the ENS. We focused our investigations on dopaminergic neurons as they are among the latest populations of neurons to differentiate during enteric development. The myenteric plexus of specific pathogen-free (SPF) and germ-free (GF) mice were examined in whole-mount preparations of the small and large intestine at three time-points: postnatal day 1 (P1), P7, and P28. The density of dopaminergic neurons did not differ significantly between SPF and GF mice in any region of the intestine examined at P1. However, at P7, GF mice had significantly fewer myenteric dopaminergic neurons in the ileum than did SPF mice, and this difference was maintained at P28.

The proportion of enteric dopaminergic neurons has been shown to be dependent upon the availability of serotonin. In the GI tract, serotonin can be of neuronal or enterochromaffin (EC) cell origin. We therefore tested the hypothesis that reductions in myenteric dopaminergic neuron densities in the ileum of GF mice were secondary to changes in enteric serotonergic neuron densities or EC cell frequencies. Neither serotonergic neurons nor EC cell numbers were affected by GF status during the postnatal period. The reduction in dopaminergic neurons seen in GF mice must therefore be attributable to a mechanism that has yet to be determined.

These findings are consistent with the notion that enteric microbiota can influence the development of late-born neuronal populations. The reduced proportion of dopaminergic neurons in the ileum of GF mice at P7 and P28 may contribute to the previously described altered motility patterns in postnatal GF mice.

abstract: Master of Science (MSc)


title: THERMALLY INDUCED WRINKLING IN MULTILAYER DECORATIVE LAMINATES AND A METHOD TO MINIMIZE

contributor:
Thompson, M. R.
David Potter, Allan Spence
Chemical Engineering

subject:
buckling
wrinkling
thermoforming
automotive decorative laminates
thin films
biaxial
in mold forming
polypropylene
pressure sensitive adhesive
lamination
Applied Mechanics
Polymer and Organic Materials
Polymer Science
Applied Mechanics

abstract: Multilayer polymer decorative films are showing a growing presence in the automotive industry, substituting conventional paint applications while maintaining similar aesthetic appeal. However for certain film constructions that have distinct layer properties, the polymer film laminates have shown to form wrinkles on application of heat during thermoforming. In this study, attempts were made to identify the factors influencing wrinkling and to predict the variation in the wrinkle parameters under changing forming conditions using existing theoretical models. A new modified thermoforming technique is proposed for producing thermoformed parts without wrinkles and thereby achieving a Class A finished surface.

abstract: Master of Applied Science (MASc)


title: Structural Analysis of DdrB from Deinococcus radiodurans: Insight into the Mechanism of Protein Mediated Single-Stranded DNA Annealing

subject:
DdrB
Deinococcus radiodurans
single-stranded DNA annealing
structural biology
Structural Biology
Structural Biology

abstract: Bacteria of the genus Deinococcus are perhaps the most resilient life forms ever discovered, demonstrating extreme resistance to ionizing radiation, ultraviolet radiation, desiccation, and a variety of mutagenic chemical agents. The most studied member of this genus, D. radiodurans, has been observed to rapidly reassemble its genome following severe fragmentation by hundreds of γ-radiation induced double-strand DNA breaks. Amongst the numerous factors contributing to DNA repair, a single-stranded annealing protein, DdrB, is believed to play an important role during the initial phases of recovery. The work described in this thesis represents the first structural characterization of DdrB, revealing a novel fold for single-stranded DNA binding. Together with biochemical data delineating the DNA-binding interface, two crystal structures of the DdrB/ssDNA complex were also solved, providing a comprehensive illustration of this interaction. Quaternary assemblies observed in these crystal structures also informed on the potential contribution of higher-order nucleoprotein complexes to the function of DdrB in single-stranded annealing. Most significantly, a face-to-face assembly of DdrB/ssDNA complexes provided insight into the mechanism by which DdrB mediates annealing of DNA, which may represent a common mechanism shared by other single-stranded annealing proteins.

abstract: Doctor of Philosophy (PhD)


title: TUMOR NECROSIS FACTOR ALPHA (TNFα) in SANDHOFF DISEASE PATHOLOGY

contributor:
Igdoura, Suleiman A.
Nurse, Colin A.
Biology

subject:
Neurodegeneration
Sandhoff disease
Lysosomal storage disease
Tumor Necrosis Factor alpha (TNF)
MicroRNA
Bone Marrow Transplantation (BMT)
Biology
Genetics
Medical Genetics
Medical Molecular Biology
Medical Neurobiology
Molecular genetics
Neurosciences
Biology

abstract:
Abstract

Sandhoff disease (SD) is a monogenic lysosomal storage disorder caused by a lack of a functional β-subunit of the beta-hexosaminidase A and B enzymes. The clinical phenotype of Hexb-/-mouse model recapitulates the symptoms and signs of Tay-Sachs and Sandhoff diseases in human. To gain insight into the neuropathology of Sandhoff disease, we defined the role of TNFα in the development and progression of Sandhoff disease pathology in mice, by generating a Hexb-/-Tnfa-/- double knock-out mouse. Behavioural testing and immunostaining data revealed the neurodegenerative role of TNFα in disease pathology. Double knock-out mice showed ameliorated clinical course, with prolonged life span. TNFα-deficient Sandhoff mice also demonstrate decreased levels of astrogliosis, and reduced neuronal cell death. Deletion of Tnfα in Sandhoff mice inhibited JAK2/STAT3 pathway, implicating its role in glia cell activation. This result points to TNFa as a potential therapeutic target to attenuate neuro-pathogenesis.

To investigate whether blood-derived or CNS-derived TNFα has the major impact on neurological function, we transplanted Hexb-/-Tnfα+/+ with bone marrow from either Hexb-/-Tnfα-/-or Hexb-/-Tnfa+/+ mice donors. Neurological tests shows a significant clinical improvement for Hexb-/-Tnfα-/- compared to Hexb-/-Tnfa+/+ recipient, regardless the genotype of donor cells. These findings highlight the importance of resident-derived TNFα during the robust neurodegenerative consequences in Sandhoff disease. To understand of the role of microRNAs in Sandhoff pathology, we investigated the miRNA profile in Sandhoff brains. A pattern of dys-regulated microRNAs was evident in Sandhoff CNS. Microarray identified miR-210 and miR-96 dys-regulated pattern in the CNS of Sandhoff mice. Strikingly, neuronal pentraxin, a putative target gene for miR-210, was induced in Sandhoff brains.

Taken together, this work establishes the proinflammatory role of TNFα in Sandhoff pathology, leading to massive neuro-apoptosis. Importantly, our studies propose that neuronal pentraxin as a novel target gene for microRNA-210 in Sandhoff brain samples, providing a potential modulator of neurodegeneration.

abstract: Doctor of Philosophy (PhD)


title: NEW APPROACH FOR ROBOTIC GRASPING OF UNKNOWN THREE DIMENSIONAL OBJECTS

contributor:
Bone, Gary M.
Capson, David
Electrical Engineering

subject:
Grasping
Vision
Robotics
Parallel Jaw
Grasp Evaluation
Laser
Robotics
Robotics

abstract: Automated grasping of objects of unknown geometry a priori has applications in many industries such as clearing a mine shaft after blasting, agricultural applications such as fruit and vegetable handling, and many roles in the service industry such as fetching items for a handicapped individual. In these roles the system environment is highly unstructured, and the robot must be able to react to different types of objects needing to be grasped. In this thesis a vision guided robotic grasp planner for unstructured environments is presented. An evaluation method for robotic grasping involving two distinct sets of objects is also presented. Both the grasp planner and evaluation metric are evaluated by experimentation using an articulated robotic arm with an eye-in-hand video camera, line laser, and pneumatic gripper. Multiple grasping experiments were performed with the objects in random poses on a modified tabletop deemed the playfield that did not allow objects to rest flat. The grasp planner focused on using a created model of the object from camera observations using silhouetting and line laser data. The object model and its computed convex hull were used to evaluate and select a single facet and point creating a grasping pair for the pneumatic gripper jaws. The grasp was attempted and then evaluated using a secondary camera and the developed evaluation method. iv Grasp success rates ranged from 80.30% (Rectangular Block on playfield 137 attempts) to 97.69% (Hexagonal Nut 173 attempts), with a mean grasp computation time for the hexagonal nut of 0.57s.

abstract: Master of Applied Science (MASc)


title: Molecular Mechanisms Regulating Ontogeny of O2- and CO2-Chemosensitivity in Rat Adrenomedullary Chromaffin Cells: Role of Nicotinic ACh and Opioid Receptor Signalling

contributor:
Nurse, Colin A.
Biology

subject:
Opioids
Nicotine
Katp channel
HIF
SIDS
Life Sciences
Medicine and Health Sciences
Life Sciences

abstract:
Catecholamine (CAT) secretion from adrenomedullary chromaffin cells (AMCs) is essential for survival of the fetus and for adaptation of the newborn to extrauterine life. CAT secretion protects the fetus from intrauterine hypoxia (low O2) and is required for maintaining cardiac conduction and preparing the lungs for air breathing. Asphyxial stressors (e.g. hypoxia, hypercapnia (high PCO2), and acidosis (low pH)) arising from labor contractions and postnatal apneas, are the main stimuli for the ‘non-neurogenic’ CAT release from perinatal AMCs. In the rat, the mechanisms of hypoxia chemosensitivity in AMCs involve inhibition of a variety of K+ channels, leading to membrane depolarization, voltage-gated Ca2+ entry, and CAT secretion. The magnitude of this depolarization is regulated by the simultaneous activation of ATP-sensitive K+ (KATP) channels, which tends to hyperpolarize the membrane potential during hypoxia. Interestingly, chemosensitivity of rat AMCs and CAT secretion in response to asphyxial stressors are markedly reduced postnatally following the development of functional innervation of these cells by the splanchnic nerve.

The primary purpose of this thesis was to delineate molecular mechanisms involved in the suppression of hypoxia and hypercapnia chemosensitivity following splanchnic innervation in neonatal rat AMCs. Experiments were designed to test the general hypothesis that the ontogeny of O2 and CO2 sensitivity in AMCs is regulated by the activation of postsynaptic nicotinic ACh and opioid receptor signalling pathways following innervation. Previous studies in this laboratory showed that exposure of perinatal rat AMCs to nicotine in utero and in vitro resulted in the selective blunting of hypoxia (but not hypercapnia) chemosensitivity. The underlying mechanism was attributable to the increased membrane hyperpolarization caused by the functional upregulation of KATP channels. In Chapter 2, I report the results of investigations of molecular mechanisms involved in the nicotine-induced upregulation of KATP channels, using a rat fetal-derived, O2- and CO2-sensitive immortalized chromaffin cell line (MAH cells), as a model. Exposure of MAH cells to chronic nicotine (50 μM) for 7 days in culture caused an increase in the expression of the KATP channel subunit, Kir6.2. This effect was blocked by α-bungarotoxin, a blocker of homomeric α7 nicotinic acetylcholine receptors (α7 nAChRs). The upregulation of Kir6.2 in MAH cells was also dependent on the transcription factor, hypoxia inducible factor (HIF)-2α. First, whereas the upregulation of Kir6.2 was present in wild type and scrambled control MAH cells, it was absent in HIF-2α-deficient (shHIF-2α) MAH cells. Second, chronic nicotine caused a progressive, time-dependent increase in HIF-2α accumulation that occurred in parallel with the increase in Kir6.2 expression. Third, chromatin immunoprecipitation (ChIP) assays revealed the binding of HIF-2α to a hypoxia response element (HRE) in the promoter region of the Kir6.2 gene. These data suggest that chronic nicotine causes the accumulation of HIF-2α which results in the transcriptional upregulation of the Kir6.2 gene. These observations were validated in an in vivo model where rat pups were exposed to nicotine in utero. Western blot analysis of adrenal gland tissues from nicotine-exposed (relative to saline-exposed) pups revealed a significant increase in Kir6.2 subunit expression and HIF-2α accumulation, and both were restricted to the medullary (but not cortical) tissue.

Chapter 3 tested the hypothesis that postnatal innervation causes the suppression of O2- and CO2-chemosensitivity in neonatal AMCs via opioid receptor signalling. It was found that chronic μ- and δ-opioid agonists (2 μM) in vitro led to the suppression of both O2- and CO2-chemosensitivity; this was correlated with the upregulation of KATP channel expression and the downregulation of carbonic anhydrase (CA) I and II respectively. The underlying molecular and signalling mechanisms were further investigated in Chapter 4. Using the MAH cell model, it was found that exposure to a combination of μ- and δ-opioid agonists for 7 days resulted in the naloxone-sensitive upregulation of Kir6.2 subunit and the downregulation of CAII. Similar to chronic nicotine exposure, the effects of chronic opioids on the upregulation of Kir6.2 and downregulation of CAII were HIF-2α-dependent. Western blot analysis revealed that HIF-2α accumulation in opioid-treated MAH cells occurred along a time-course that paralleled the upregulation of Kir6.2 subunit. ChIP assays demonstrated the binding of HIF-2α to the promoter region of the Kir6.2 subunit gene in opioid-treated MAH cells. Moreover, PKA activity (but not PKC or CaMK) was found to be required for the effects of opioids on Kir6.2 and CAII expression, but not HIF-2α accumulation. In complementary in vivo studies, adrenomedullary tissues from morphine-exposed rat pups showed an increased expression of both HIF-2α and Kir6.2, and decreased expression of CA1 and II protein. These findings have uncovered novel mechanisms by which postnatal innervation contributes to the ontogeny of O2- and CO2-chemosensitivity in rat adrenal chromaffin cells. They also suggest mechanisms by which exposure of the fetus to nicotine in cigarette smoke or opioids from drug abuse might contribute to abnormal arousal reflexes, and pathophysiological conditions such as Sudden Infant Death Syndrome (SIDS).

abstract: Doctor of Philosophy (PhD)


title: Comprehensive Metabolomic Analysis in Peanut Sensitization and Peanut-Induced Anaphylaxis: Discovery of Biomarkers and Mediators

contributor:
Jordana, Manel
Mark Larché and Susan Waserman
Medical Sciences

subject:
Peanut Allergy
Anaphylaxis
Metabolomics
Sensitization
Uric Acid
Animal Model
Medical Immunology
Medical Pathology
Medical Immunology

abstract:
BACKGROUND: The ontogeny of peanut allergy (PA) is poorly understood, and the treatment of its most severe manifestation, peanut-induced anaphylaxis (PIA), remains limited to rescue epinephrine. We argued that an untargeted metabolomic analysis would be a useful hypothesis-generating tool to identify novel biomarkers, mediators and possibly therapeutic targets in PA and PIA.

METHODS: Models of PA and PIA used in this thesis involved either the oral administration of peanut along with cholera toxin or the topical application of peanut on tape-stripped skin. Liquid-chromatography mass-spectrometry (LC-MS) was performed to identify chemical changes in the serum of mice undergoing sensitization and anaphylaxis. Flow cytometry as well as in vivo gain-of-function and loss-of-function immunological studies were used to determine the biological significance of particular molecules in sensitization.

RESULTS: LC-MS followed by multivariant analysis showed that the purine metabolism pathway was altered with elevated levels of uric acid (UA) in sensitized mice. UA depletion using allopurinol and uricase fully prevented the development of the allergic and anaphylactic phenotype. Conversely, administration of UA crystals, instead of cholera toxin or tape stripping along with peanut induced a typical allergic and anaphylactic phenotype. The effects of UA and UA crystals are likely a consequence of effects on the activation of resident dendritic cells. Post-challenge metabolic analysis also revealed a distinct metabolic signature in sensitized mice, highlighted by an increase in several metabolites such as histamine. Likewise, peanut allergic patients display a distinct metabolic profile after oral peanut challenge.

CONCLUSION: We identified UA, released after damage to the mucosa and/or skin, as a critical alarmin that facilitates the development of Th2 immunity, specifically PA and PIA. Metabolomics analyses of either mice undergoing anaphylaxis or peanut allergic children subjected to a peanut oral challenge provided an extensive overview of metabolomic changes underlying these conditions. Further studies may lead to the identification of novel biomarkers and mediators.

abstract: Master of Science in Medical Sciences (MSMS)


title: “Never Say DIE!” An Ethnographic Epidemiology of Helicobacter pylori Infection and Risk Perceptions in Aklavik, NWT

contributor:
Herring, D. Ann
Warry, Wayne
Young, Kue T.
Anthropology

subject:
ethnography
epidemiology
Arctic
H. pylori
stomach cancer
Indigenous health
Other Anthropology
Other Anthropology

abstract:
Helicobacter pylori is a bacterial infection of the stomach lining known to cause ulcers and stomach cancer This infection has become a major concern of Indigenous peoples living in the Northwest Territories, where H. pylori infection and stomach cancer are more prevalent relative to much of southern Canada and the United States. I joined the Canadian North Helicobacter pylori (CANHelp) Working Group in 2010 to conduct participant observation in the Aklavik H. pylori Project (AHPP) and identify ways that ethnography can be integrated into the ongoing multi-pronged research that incorporates epidemiology, microbiology, gastroenterology, knowledge translation, and the development of public health policy.

Between September, 2011 and June, 2012, I lived as a participant observer in Aklavik. I led an epidemiological study of the incidence and re-infection of H. pylori infection. I examined how different risk perceptions emerge from processes of “making sense” of H. pylori as a “pathogen” or as a “contaminant” and described how these different constructions influence people’s behaviours. Ethnography, in this way, can make visible the lenses through which different groups of actors perceive, experience, and react to H. pylori infection. The recognition that the social inequities most strongly associated with H. pylori infection and re-infection that exist today are the result of Aklavik’s colonial history is one example of a space in which different lenses can be brought into a shared focus. From such shared understandings, consensus knowledge can be built collaboratively between outside researchers and Indigenous Arctic communities in an ongoing, and community-driven, research project. Furthermore, I critically examined the definition and use of the “household” as a level-unit of risk assessment and have outlined steps for assessing possible risk factors as these are distributed across multi-household extended kin groups that can be identified and followed in long-term research.

abstract: Doctor of Social Science


title: CHARACTERIZATION OF A POPULATION OF TUMOUR-INITIATING CELLS WITH STEM-LIKE PROPERTIES IN HUMAN PROSTATE CANCER

contributor:
Tang, Damu
Medical Sciences

subject:
Prostate cancer stem cell
Self-renewal
SOX2
EGFR
MEK-ERK (MAPK) pathway
PI3K-AKT signalling and PTEN
Medical Molecular Biology
Medical Molecular Biology

abstract:
There is increasing evidence that prostate tumours are organized as a hierarchy with rare cancer stem cells (CSCs) implicated in initiating and maintaining the tumour. However, prospective prostate cancer stem cells (PCSCs) have not been thoroughly characterized from primary tissue specimens. Using the DU145 cell line, PCSCs have been propagated as non-adherent spheres in vitro. Approximately 1.25% of monolayer DU145 cells formed primary spheres while 26% of sphere cells formed subsequent spheres; a measure of PCSC self-renewal capacity. Spheres are enriched for cells expressing prostate basal and luminal cytokeratins and CSC markers (CD44, CD24, integrin alpha2beta1). PCSCs initiate xenograft tumours with enhanced capacity compared to monolayer cells. While epidermal growth factor (EGF) promoted PCSC propagation, basic fibroblast growth factor (bFGF) inhibited these events. Activation of EGF receptor (EGFR) signalling, following EGF treatment or expression of constitutively-active EGFR (EGFRvIII), increased sphere formation. Conversely, attenuation of EGFR signalling inhibited PCSC self-renewal. Consistent with the MEK-ERK pathway being a major target of EGFR signalling, the MEK-ERK pathway contributes to EGFR-facilitated PCSC propagation. Inhibition of ERK activation following MEK inhibitor treatment, expression of dominant-negative MEK1(K97M), or knockdown of ERK1 or ERK2 reduced PCSC propagation. Therefore, EGFR signalling promotes PCSC self-renewal by activating the MEK-ERK pathway.

SOX2 is an essential transcription factor for stem cells, however, its role in PCSCs remains unclear. SOX2 protein is upregulated in PCSCs propagated as spheres, and its expression is regulated by EGFR signalling. EGFR activation, following EGF treatment or expression of constitutively-active EGFRvIII, increased SOX2 expression and PCSC self-renewal, while being attenuated by EGFR inhibitor treatment. Ectopic SOX2 expression enhanced EGF-induced PCSC self-renewal, while SOX2 knockdown renders PCSCs non-responsive to EGF-induced self-renewal and reduced their anchorage-independent growth. Furthermore, SOX2 expression is associated with the ability of PCSCs to form aggressive xenograft tumours. Collectively, SOX2 regulates EGFR-mediated PCSC self-renewal.

abstract: Doctor of Philosophy (PhD)


title: Genetic Manipulation of Secondary Metabolite Production in Actinomycetes

contributor:
Nodwell, Justin
Brian Coombes and Marie Elliot
Biochemistry and Biomedical Sciences

subject:
Actinomycetes
Streptomyces
Antibiotics
Regulation
Secondary metabolite production
Biochemistry
Biochemistry

abstract: The world is facing a public health threat due to increasing emergence of antibiotic resistance in pathogens. Streptomyces the soil-dwelling, Gram-positive, filamentous bacteria belonging to the family actinomycetes, are proven to be rich sources of natural antibiotics. Genome sequencing of Streptomyces coelicolor, a model organism of this genus, has revealed that in addition to the five antibiotics characterized so far, it possesses abundant genetic architecture of unexpressed biosynthetic or cryptic clusters for secondary metabolite production. The reason for their silence appears to be the poor understanding of their specific activation stimuli. In Streptomyces coelicolor, a pleiotropic regulator belonging to the two-component system family, afsQ1, has shown to activate the production of actinorhodin (ACT), undecylprodigiosin (RED), and calcium-dependent antibiotic (CDA). The aim of this research was to employ the genetically engineered afsQ1 allele (named afsQ1*), which mimics the phosphorylated active form and obviates the need for specific external stimulus, and screen for novel antibiotic production. In this study, afsQ1* was introduced in various wild actinomycete isolates from the Wright Actinomycetes Collection (WAC) by conjugation and the resulting mutants were screened for antibiotic production. Two out of six WAC strains showed afsQ1*- induced antimicrobial activity. Interestingly, we were able to purify two antibiotic compounds, namely 1082 [M+2H]2+ and 782 [M+H]+ from the strain WAC00263. 1082 [M+2H]2+, a potentially novel antimicrobial peptide, exhibited activity against a wide range of Gram-positive bacteria including resistant pathogens such as vancomycin-resistant Enterococcus ATCC# 51299, a clinical isolate of methicillin resistant Staphylococcus aureus, and a clinical isolate of S. aureus BM3002. Moreover, it also showed activity against an opportunistic Gram-negative multi-drug resistant pathogen Acinetobacter baumannii B0098426R and a virulent strain of the fungus Cryptococcus neoformans H99. The second newly expressed molecule, 782 [M+H]+ was not as potent as 1082 [M+2H]2+, so far only exhibited antimicrobial activity against the Gram-positive laboratory strains Bacillus subtilis #168 and Micrococcus luteus. These results reiterate that the technique of heterologous expression of the pleiotropic regulator, afsQ1*, in diverse actinomycetes is an excellent tool to induce novel antimicrobial production.

abstract: Master of Science (MSc)


title: STAT3 and SMAD Signaling in Mouse Models of Oncostatin M-Induced Lung Extracellular Matrix Remodeling

contributor: Medical Sciences (Molecular Virology and Immunology Program)

subject:
Oncostatin M
lung fibrosis
bone morphogenetic protein
transforming growth factor beta
fibroblast
airway epithelial cells
Circulatory and Respiratory Physiology
Circulatory and Respiratory Physiology

abstract:
IPF is a respiratory condition of unknown etiology that has poor survival prognosis. The stiffening of the lung associated with this condition is attributed to the irreversible turnover of healthy lung tissue into scar tissue, which affects gas exchange and can eventually lead to organ failure. Numerous studies have implicated the pro-fibrogenic growth factor TGF-β, through activation of the SMAD2/3 pathway, as a central mediator in the pathology of this condition. However, other cytokines, including members of the IL-6/gp130 family such as OSM, and other signaling pathways may be implicated in ECM accumulation in certain conditions. In particular, STAT3 activation and an impairment of the BMP-SMAD1 signaling axis is thought to contribute to lung ECM accumulation. Based on the finding that transient pulmonary overexpression of OSM induces lung ECM accumulation in C57Bl/6 mice, it was hypothesized that OSM-induced ECM remodeling would be associated with STAT3 activation and suppression of the BMP-SMAD1-signaling axis.

Findings in this thesis revealed that transient pulmonary overexpression of OSM induces ECM remodeling in both BALB/c and C57Bl/6 mice after seven days, despite a dichotomous response in other experimental models of ECM remodeling. However, parenchyma, but not airway, pathology resolved after 28 days in AdOSM-treated BALB/c mice. Furthermore, OSM-induced ECM remodeling occurred independently of IL-6-associated inflammation as well as TGF-β/SMAD3 signaling. MLF cultures treated with OSM did not directly regulate gene expression of ECM-related genes, suggesting that other cells may be responsible for OSM-induced ECM accumulation in vivo. OSM overexpression in vivo was associated with STAT3 activation and SMAD1 suppression, and an assessment of STAT3 and SMAD signaling in vitro showed that OSM activated the STAT3 pathway in MLF cultures, mouse type two pneumocytes, and human airway cells, while OSM suppressed the SMAD1 pathway in mouse type two pneumocytes, and human airway cells. Collectively, this thesis shows that OSM induces novel pathways in models of lung ECM remodeling, and this may have implications for IPF pathogenesis.

abstract: Master of Science (MSc)


title: DESIGN OF ASYMMETRIC REFLECTIVE SEMICONDUCTOR OPTICAL AMPLIFIER IN WAVELENGTH DIVISION MULTIPLEXING PASSIVE OPTICAL NETWORKS

contributor:
Li, Xun
Electrical and Computer Engineering

subject:
Asymmetric RSOA
WDM-PON
Electrical and Computer Engineering
Electrical and Computer Engineering

abstract:
Reflective semiconductor optical amplifiers (RSOA) are widely used in wavelength-division-multiplexed passive optical networks (WDM-PON). RSOAs in optical network units (ONUs) are operated in the gain saturation region so that the amplitude squeezing effect can be used to erase the information on downstream signals, however, the upstream signals go through the same RSOA need to be amplified.

In order to use one RSOA to satisfy the need of erasing the information on downstream signal by using amplitude squeezing effect and amplifying the upstream signal at the same time, an asymmetric RSOA design is proposed and demonstrated in this thesis. The ridge width becomes narrower along the traveling direction of the downstream signal so that the downstream signal can be amplified in the saturation region of the RSOA. At the same time, the ridge width increases in the traveling direction of the upstream signal, so that the modulated upstream signal can be amplified by the asymmetric RSOA. In this thesis, I mainly focus on the designing of the structure of the RSOA to enlarge the gain difference between upstream and downstream gain. Difference between wide end and narrow end effective indices, cavity length, the way that effective index changes from the wide end to the narrow end and bias current are factors that can affect the gain difference. How the device performance is affected by the factors were analyzed. An optimized structure of asymmetric RSOA is then proposed according to the effects of the factors. The performance of the asymmetric RSOA, including gain dynamic performance, saturation output power, and upstream output power, is compared with symmetric RSOA. The asymmetric RSOA shows better performance in erasing the downstream signal information as well as amplifying upstream signal.

abstract: Master of Applied Science (MASc)


title: Tissue Stabilization in MRI-Guided Breast Biopsy

contributor:
Sirouspour, Shahin
Patriciu, Alexandru
Jeremic, Aleksandar
Biomedical Engineering

subject: Breast biopsy

subject: Magnetic Resonance Imaging

subject: Tissue stabilization

subject: MR-compatibility

subject: MR-guided breast biopsy

subject: Compression plates

subject: Biomedical Engineering and Bioengineering

abstract:
Breast cancer is the most common form of cancer in women in the United States. Histopathological examination through breast biopsy is considered as the "Gold Standard" for a definitive diagnosis. Contrast-enhanced Magnetic Resonance Imaging (MRI) is often used for guiding the biopsy in those cases in which the tumor may not be detectable under Ultrasound or X-ray mammography. Stabilization of the breast tissue during the biopsy is critical for its success to ensure that the target would not be displaced due to the patient movement or tissue deformation. Conventionally, the breast tissue is immobilized by firmly compressing it between two parallel plates. However, high compression forces causes significant patient discomfort and can reduce the intake of the contrast agent, which negatively impact the image quality.

This thesis introduces devices and control methodologies for active tissue stabilization in magnetic resonance imaging (MRI)-guided breast biopsy. Pneumatic and piezoelectric actuators have been considered for developing concept designs for MRI-compatible tissue stabilization devices. Only the pneumatic device has been prototyped and tested. The device is comprised of two pneumatically-actuated support plates that would stabilize the biopsy target movements during needle insertion. An optimized geometry for the support plates allows for a good degree of tissue stabilization without relying on large compression forces. The plate configuration can also be adjusted inside the magnet bore using pneumatic actuators driven by pressure-controlled valves that are placed in the MR control room. This capability allows for the compensation of the target displacement based on MR image feedback. When combined with a separate needle drive mechanism, this stabilization device would enable in-bore MR-guided breast biopsy in combination with an in-bore needle driver system. The proposed approach offers improved target stabilization at reduced compression force and patient discomfort, that may also enhance MR image quality as result of greater intake of contrast agent. The open-front design of the stabilization plates provides greater flexibility in selecting the needle insertion entry point, and active adjustment of the support plates based on MR feedback improves the targeting accuracy.

A concept design for a MR-compatible needle driver mechanism using piezoelectric actuators is also proposed. Experiments performed on chicken breast tissue with a prototype of the device demonstrate the effectiveness of this mechanism in increasing needle targeting accuracy using two simple error correction strategies. Furthermore, MRI compatibility tests are carried out to asses the performance of the device inside MRI.

abstract: Master of Applied Science (MASc)


title: Electrodeposition of Tunable Zinc Oxide Nanomaterials for Optical Applications

contributor:
Soleymani, Leyla
Engineering Physics

subject: Electrodeposition of zinc oxide (ZnO)

subject: Motheye anti-reflection coatings

subject: zinc oxide nanomaterials

subject: anti-reflection coatings

subject: solar optical applications

subject: photovoltaics

subject: Engineering Science and Materials

subject: Materials Science and Engineering

subject: Operations Research, Systems Engineering and Industrial Engineering

subject: Engineering Science and Materials

abstract:
Renewable energy technologies and the development of cleaner and more environmentally friendly power have been at the forefront of research for the past few decades. Photovoltaic systems – systems that convert photon energy to electrical energy – are at the center of these research efforts. Decreasing the cost of energy production, through increasing the power conversion efficiency or decreasing the device cost, is a key factor in widespread use of these energy production systems. To increase the energy conversion efficiency, ideally, all useful photons should be absorbed by the solar cell; however, due to the large discontinuity in the refractive index at the solar cell/air interface, a large fraction of incidence light is lost due to reflection (30% loss in crystalline silicon cells). The currently used single and double layer anti-reflection coatings reduce the reflection losses, but their optimal performance is limited to a narrow range of wavelengths and angles of incidence. Moth-eye anti-reflection coatings are composed of patterned single layer films having a gradual decrease in refractive index from the solar cell surface to air. This study is focused on developing an inexpensive method for direct deposition of patterned films – in the form of moth-eye anti-reflection coatings – on solar cell surface.

In this research, the creation of moth-eye anti-reflection coatings has been attempted through the process of electrodeposition. ZnO was chosen for the thin film material, and the ability to develop the required moth-eye structure by changing the electrodeposition parameters including temperature, applied potential, type and concentration of solution-borne species, and type of substrate was investigated. Using this method, pyramidal and hemispherical structures with a 100-200 nm diameter and 100-200 nm height were created directly on ITO substrates. Similar structures were also developed on silicon substrates. The anti-reflection properties of ZnO-coated silicon substrates were investigated by comparing their broadband and broad angle reflection-mode UV-VIS spectrum with uncoated silicon. The optimized ZnO-coated silicon substrate showed a reflectance of at most 20% for wavelengths between 400-1500 nm at angles of incidence less than 50O.

abstract: Master of Applied Science (MASc)


title: Elucidating the Role of Tcf7 Isoforms in Mouse Embryonic Stem Cell Self-Renewal and Differentiation

contributor:
Doble, Bradley W.
Kristin Hope, Darren Bridgewater
Biochemistry and Biomedical Sciences

subject:
Wnt signaling
Tcf/Lef
Embryonic stem cell
TALEN technology
Gene targeting
Biochemistry
Biochemistry

abstract:
Recent advances in gene targeting technology have significantly shaped modern-day mouse genetics, as they allow for the accurate analysis of gene function in vivo. By capitalizing on conventional methodologies that are based on homologous recombination, the advent of artificially engineered nucleases, like transcription activator-like effector nucleases (TALENs), enables precise genome editing without the need for conventional targeting vectors, which typically possess long “arms” of homology that are difficult to work with, even with recombineering strategies employing bacterial artificial chromosomes. Unlike traditional techniques, these novel nucleases can be engineered in less than a week and together with compact targeting vectors, can be used to easily manipulate almost any locus in the mouse genome.

The current selection of commercially available antibodies makes it difficult to assess the specific roles of protein isoforms during early development. The Tcf/Lef family of transcription factors comprise of key downstream effector proteins of the canonical Wnt/β-catenin signal transduction cascade. This pathway is implicated in the regulation of self-renewal and is dysregulated in a number of human diseases including cancers. Among the Tcf/Lef factors, Tcf3 has been heavily studied in mouse embryonic stem cells, due at least in part to the observation that its transcript levels are expressed at the highest levels compared to the others. Recently, it was proposed that a switch takes place between a repressive state mediated by Tcf3 to an activating β-catenin-Tcf1 complex in response to Wnt signals. Here, we use TALEN technology to introduce an epitope tag at the endogenous locus of mTcf7, the gene encoding the Tcf1 protein. By tagging the N-terminus of full-length and N-terminally truncated dominant-negative variants of Tcf1, we establish a tool to better study a previously unappreciated role for Tcf1 in regulating embryonic stem cell self-renewal and differentiation. Furthermore, we also show that the tagged variants generated exhibit similar protein expression levels to those of wild-type controls, and display nuclear localization as expected.

abstract: Master of Science (MSc)


title: Fault Detection and Diagnosis of a Diesel Engine Valve Train

contributor:
Bone, Gary M.
Veldhuis
Mechanical Engineering

subject:
Fault Detection
Signal Processing
Vibration
Signal Monitoring
Automotive
Diesel Engine
Acoustics, Dynamics, and Controls
Mechanical Engineering
Signal Processing
Statistical Models
Acoustics, Dynamics, and Controls

abstract: One of the most commonly used mechanical systems is the internal combustion engine. Internal combustion engines dominate the automotive industry, and have numerous other applications in generation, transportation, etc. This thesis presents the development of a fault detection and diagnosis (FDD) system for use with an internal combustion engine valve train. A FDD system was developed with a focus on the valve impact amplitudes. Engine cycle averaging and band-pass filtering methods were tuned and utilized for improving the signal to noise ratio. A novel feature extraction method was developed that included a local RMS sliding window method and an adaptive threshold. Faults were seeded in the form of deformed valve springs, as well as abnormal valve clearances. The engine’s manufacturer specifies that a valve spring with 3 mm or more of deformation should be replaced. This thesis investigated the detection of a relatively small 0.5mm spring deformation. Valve clearance values were adjusted 0.1mm above and below the nominal clearance value (0.15mm) to test large clearance faults (0.25mm) and small clearance faults (0.05mm). The performance of the FDD system was tested using an instrumented diesel engine test bed. A comparison of numerous signal processing techniques and classification methods was performed.

abstract: Master of Applied Science (MASc)


title: THE ROLE OF IL-15 AND NK CELLS IN BREAST TUMOR FORMATION AND METASTASIS

contributor:
Ashkar, Ali
Medical Sciences

subject:
breast cancer
NK cells
IL-15
immunotherapy
metastasis
Medical Sciences
Medical Sciences

abstract: IL-15 is a cytokine that has effects on both innate and adaptive immune cells, including NK and CD8 T cells. The involvement of these cell types in tumor immunosurveillance and eradication has led to interest in IL-15 as an immunotherapy. Its role in spontaneous solid cancers has not been studied thoroughly. Here, we have shown for the first time that IL-15 overexpression in a spontaneous breast cancer model, MMTV-polyoma Middle T antigen (MT), leads to increased survival, tumor destruction, and decreased metastasis (IL-15 TG/MT). In contrast, lack of IL-15 led to decreased survival (IL-15 KO/MT) and increased metastasis. Protection in IL-15 TG/MT mice was dependent upon the presence of highly activated NK1.1+ cells, but not dependent upon CD8 T cells. The cytokine environment in IL-15 TG/MT tumors was capable of activating human NK cells to kill human triple negative breast cancer cells. In a model of metastasis, we found that overexpression of IL-15 protected from metastasis in a NK cell dependent manner. Lack of IL-15 promoted the polarization of CD4 T cells to a Th2 phenotype and they influenced polarization of macrophages to an M2 phenotype. M2 macrophages help establish tumors at the metastatic site. Here we found that M1 polarized macrophages could prevent engrafted breast tumor formation, whereas M2 macrophages promoted it. Overall, IL-15 is an extremely promising immunotherapy that has more anti-tumor effects on the immune system than were previously appreciated. Additionally, our data argues that it could generate immune responses against both primary breast cancer and metastasis.

abstract: Doctor of Philosophy (Medical Science)


title: SOLVENT FREE EMULSIFICATION IN A TWIN-SCREW EXTRUDER

contributor:
Thompson, Michael
Chemical Engineering

subject:
Latex
Solvent-free
extruder
emulsification
residence time
morphology
Other Chemical Engineering
Other Chemical Engineering

abstract:
The production of latex in a solvent-free process within a twin screw extruder is of great industrial interest given the associated reduction in environmental impact when compared to conventional solvent-based emulsification techniques. The ability to produce latex continuously is also advantageous, compared to batch-wise solvent-based processes.

The process of solvent-free emulsification in a twin screw extruder is studied. An inline fiber optic spectroscopic system was installed in the extruder to study the residence time distribution profile of resin through the emulsification process. A design of experiment study was performed analyzing the response of latex particle size from screw speed and feed rate factors. Finally, scanning electron microscopy was used to determine the morphology of the poorly- emulsified and pre-emulsified resin.

Experimental results demonstrate that the residence time distribution of the polymer within the extruder is largely invariant of screw speed; the system also demonstrated a very low degree of axial mixing – which was not expected but can be attributed to high degree-of-fill within the screw. The results of the design of experiments study show that the final latex particle size is more influenced by the feed rate of the polymer than the screw speed of the extruder. These results were found to be consistent with the literature based on both batch- phase inversion emulsification as well as continuous polymer blending of immiscible phases. Finally, results from a study on morphology provided evidence of water domains within the pre-inverted polymer domains, analogous to a ‘water-in-oil-in water’ dispersion. Evidence of a bicontinuous network of polymer and water was also found to exist. The summation of these experimental results leads to the hypothesis that the solvent-free emulsification process is a phase inversion-type emulsification mechanism as opposed to a direct emulsification mechanism.

abstract: Master of Applied Science (MASc)


title: MODELING AND CONTROL OF AN IMPROVED HYBRID PNEUMATIC-ELECTRIC ACTUATOR

contributor:
Bone, Gary M.
Veldhuis, Sirouspour
Mechanical Engineering

subject:
Actuators
pneumatic actuators
hybrid actuators
position control
robot control
Controls and Control Theory
Electro-Mechanical Systems
Controls and Control Theory

abstract: Combining the advantages from electric motor and the pneumatic actuator, the hybrid pneumatic-electric actuator is expected to be safe, low-cost, clean, high power to weight ratio, and to provide precise position control. In this thesis, the modeling and control of an improved hybrid pneumatic-electric actuator prototype is presented. The actuator’s main components consist of a low-friction pneumatic cylinder, two on/off solenoid valves, and a small DC motor. The cylinder and motor are connected to a common output shaft using gears. The shaft rotates a single-link robot arm. Its position is measured by an incremental encoder. The prototype was improved by incorporating faster switching valves, flow controls, a faster valve drive circuit, a high resolution encoder rather than the existing linear potentiometer, more accurate pressure sensors and stronger gears. A system dynamic model without the valve dynamic was developed identified and validated using open-loop experiments. The valve models for a discrete input and PWM input were then developed and validated separately. The use of bipolynomial function and artificial neural network fitting methods for modeling the valve mass flow rates were compared. The combined system model with valve dynamics was validated experimentally. Two model-based nonlinear position controllers, using the backstepping and discrete-valued model predictive control (DVMPC) methods, were designed, simulated and extensively tested. Testing was done with the actuator operating using the cylinder alone, the motor alone and in hybrid mode using the cylinder and motor together. Operating in the hybrid mode reduced the root-mean-square error (RMSE) up to 80%. A stability analysis for the backstepping control including the valve modeling error, friction model error, and electric motor torque modeling error was performed. Compensation terms were designed to improve the performance for the two controllers. Additional stability analyses were performed for backstepping controller with a feedback term and the DVMPC with motor control. A payload estimation algorithm was proposed and shown to enhance the robustness of the DVMPC operating in vertical configuration. Simulations and experiments demonstrated that the model-based controllers performed well for both vertical and horizontal configurations. Regarding robustness to payload mismatch, if the payload was within the load capacity of the hybrid actuator, the model-based controllers were both insensitive to the payload variations in horizontal configuration. The backstepping controller was also robust to the payload variations in the vertical configuration. In experiments, the backstepping control in hybrid actuation mode produced a RMSE of 0.0066 radian for a 2 Hz sine wave desired position trajectory with a 0.3 radian amplitude. With DVMPC, this value decreased to 0.0045 radian. These tracking errors were shown to be 30 to 50% less than those produced by a modified linear position plus velocity plus acceleration controller.

abstract: Master of Applied Science (MASc)


title: CHARACTERIZATION OF CYB5D2 AND ITS HEME BINDING ASSOCIATED FUNCTIONS

subject:
cytochrome P450 reductase
CYP51A1
SREBP
Insig1
cholesterol
chemotherapeutic survival
Medical Sciences
Medical Sciences

abstract: Cytochrome b5 heme binding domain 2 (CYB5D2) is a heme binding protein that was initially identified for its ability to attenuate the function of the PTEN tumor suppressor gene. CYB5D2 sustains ectopic PTEN expression in U87 cells, and can also confer survival from serum starvation in NIH3T3 cells. An antibody was generated to the carboxyl terminus of CYB5D2 to detect endogenous protein expression. The highest expression of CYB5D2 protein is in neural cancer cell lines. CYB5D2 is weakly expressed in breast and kidney cancer cell lines, and moderately expressed in prostate cancer cell lines. To investigate the role of the heme binding domain in CYB5D2, a conserved aspartic acid (D86) within this domain was mutated to glycine, and this was characterized as being unable to bind heme. CYB5D2(D86G) displayed a loss of function compared to wild-type CYB5D2. To study the loss of expression of CYB5D2, stable CYB5D2 shRNA was achieved in HeLa and Huh7 cells. While ectopic CYB5D2 inhibited HeLa cell proliferation and growth in soft agar, CYB5D2(D86G) expression and CYB5D2 shRNA increased cell proliferation and soft agar growth. While ectopic CYB5D2 conferred survival from chemotherapeutic drugs in HeLa cells, CYB5D2(D86G) and CYB5D2 shRNA cells were susceptible to drug treatments. CYB5D2 inhibits SREBP signalling, which requires its heme binding ability. Using cyclohexamide treatments, CYB5D2 stabilized ectopic Insig1, while CYB5D2(D86G) destabilized ectopic Insig1. CYB5D2 shRNA reduced endogeneous CYP51A1 (lanosterol demethylase) and Insig1 protein levels, and increased the susceptibility of HeLa cells to mevalonate treatments. Furthermore, CYB5D2 shRNA HeLa cells displayed reduced CYP3A4 activity, a cytochrome P450 enzyme involved in drug metabolism. CYB5D2 binds to cytochrome P450 reductase (POR), while CYB5D2(D86G) cannot. CYB5D2 co-immunoprecipitates with endogenous POR under serum-free conditions in HeLa and Huh7 cells, while CYB5D2(D86G) cannot. Collectively, CYB5D2 is a POR interacting protein, which regulates CYP51A1 and CYP3A4 activity.

abstract: Doctor of Philosophy (Medical Science)


title: THE INFLUENCE OF THE ADAPTIVE IMMUNE SYSTEM ON BEHAVIOUR, BEHAVIOURAL SYSTEMS, AND FUNCTIONAL NEUROANATOMY

contributor:
Foster, Jane A.
Neuroscience

subject:
Neuroscience
Neuroimmunology
Anxiety
Fear
Stress
Neuronal Gene Expression
Immune Challenge
Behavioral Neurobiology
Molecular and Cellular Neuroscience
Behavioral Neurobiology

abstract: Immune-brain communication has important influences on stress circuitry and stress-related behaviours. Adaptive immune deficiency through loss of lymphocytes or an absence of gut bacteria has been linked to anxiety behaviours and stress responsiveness. In these models, there is a common deficit of T lymphocytes leading to the central hypothesis that T lymphocytes influence stress responsiveness and stress-related behaviours. This project considers the effects of T lymphocyte deficiency on anxiety and fear related behaviours as well as stress responsiveness in the hypothalamic pituitary adrenal (HPA) axis. Mice lacking T lymphocytes through knockout of the T cell receptor (TCR) β and δ chains, and B lymphocytes through knockout of the immunoglobulin M μ chain, were obtained and compared to C57BL/6 control mice. Activity, exploration, anxiety, fear and spatial learning tests were employed. Separately, gene expression was assessed for genes related to stress circuitry following chronic restraint stress. Additionally, lipopolysaccharide was used to determine the stress response to an innate immune challenge that was previously shown to elicit an exaggerated stress response in mice lacking Class I Major Histocompatibility Complex (MHC) and CD8+ T lymphocytes. It was found that mice lacking T lymphocytes, but not B lymphocytes, have reduced anxiety-like behaviour but an increased fear response. TCRβ-/-δ-/- mice also had altered expression of components of the HPA axis, serotonergic receptors and NMDA receptor subunits indicating an altered response to chronic stress. Finally, TCRβ-/-δ-/- mice do not display an exaggerated stress response to an innate immune challenge suggesting a central role for Class I MHC in the stress response that is not due to the CD8+ T lymphocyte deficiency that accompanies the functional loss of Class I MHC. These studies reflect an important role for T lymphocytes specifically in the development of the stress system and stress-related behaviours and enables a deeper understanding of neuroimmune influences on stress.

abstract: Doctor of Philosophy (PhD)


title: THE EFFECTS OF LONG-TERM WATER TABLE MANIPULATIONS ON PEATLAND EVAPOTRANSPIRATION, SOIL PHYSICAL PROPERTIES, AND MOISTURE STRESS

contributor:
Waddington, James Michael
Geography and Earth Sciences

subject:
eddy covariance
porometry
stochastic modelling
southern boreal peatland
Hydrology
Hydrology

abstract:
Northern boreal peatlands represent a globally significant carbon pool that are at risk of drying through land-use change and projected future climate change. The current ecohydrological conceptualization of peatland response to persistent water table (WT) drawdown is largely based on short-term manipulation experiments, but where the long-term response may be mediated by vegetation and microtopography dynamics. The objective of this thesis is to examine the changes to peatland evapotranspiration, soil physical properties, and moisture stress in response to a long-term WT manipulation. The energy balance, hydrology, vegetation, and soil properties were examined at three adjacent peatland sites in the southern sub-boreal region which were subjected to WT manipulations on the order of ±10 cm at two treatment sites (WET, and DRY) compared to the reference site (INT) as a result of berm construction in the 1950s.

Sites with an increasing depth to WT were found to have greater microtopographic variation and proportion of the surface covered by raised hummocks. While total abundance of the major plant functional groups was altered, species composition and dominant species of vascular and non-vascular species within microforms was unaltered. Changes in vegetation and microtopography lead to differences in albedo, surface roughness, and surface moisture variability. However, total ET was only significantly different at the WET site. Transpiration losses accounted for the majority of ET, where LAI best explained differences in total ET between sites. Surface moisture availability did not appear to be limiting on moss evaporation, where lab results showed similar moisture retention capacity between microforms and sites, and where low surface bulk density was shown to be a strong controlling factor. Modelling results further suggested that, despite dry surface conditions, surface moisture availability for evaporation was often not limited based on several different parameterizations of peat hydraulic structure with depth.

abstract: Doctor of Philosophy (PhD)


title: Single Event Upset error detection on routing tracks of Xilinx FPGAs

contributor:
Lawford, Mark
Nicolici, Nicola
Down, Douglas
Computing and Software

subject: FPGA

subject: SEU

subject: Xilinx

subject: routing

subject: switchbox

subject: error detection

subject: VLSI and circuits, Embedded and Hardware Systems

abstract: This thesis proposes a new method to detect routing switch alterations on FPGAs in real-time. By sampling the circuit path at the source and destination, and comparing the samples, it is possible to find out if there has been a routing change in the circuit path. We compare and contrast this probing method with previously established techniques such as Cyclic Redundancy Checks, Built-in-self-tests, Triple Modular Redundancy, Duplication with Comparison, and redesigning the FPGA. The probe method finds the routing error in one clock cycle, using the pre-existing elements on the FPGA, while the FPGA is still operational. This method works on all FPGAs that use the Wilton style switchbox. An automated tool for probing the design circuit is presented in this thesis that applies the probe scheme on a circuit built on the Xilinx Virtex-4 FPGA.

abstract: Master of Applied Science (MASc)


title: THE DEVELOPMENT AND OPTIMIZATION OF A HUMAN MEGAKARYOCYTE CULTURE FROM HEMATOPOIETIC PROGENITOR CELLS ISOLATED FROM NORMAL PERIPHERAL BLOOD FOR IN VITRO INVESTIGATION OF PLATELET DISORDERS

contributor:
Nazi, Ishac
Arnold, Donald M.
Truant, Ray
Biochemistry and Biomedical Sciences

subject:
Megakaryocyte culture
megakaryopoiesis
platelets
thrombopoietin
ITP
autoantibody
hematopoietic progenitor cells
peripheral blood
polyploidy
Medical Cell Biology
Medical Cell Biology

abstract:
Megakaryocyte cultures are a strong tool for the in vitro investigation of platelet production in platelet disorders. Peripheral blood derived hematopoietic progenitor cells (PB-HPCs) are the most accessible source of HPCs with high potential to produce mature megakaryocytes in vitro; however, they are present in low numbers making peripheral blood an inefficient source. Additionally, a megakaryocyte culture with an optimized thrombopoietin (TPO) concentration is required which can reliably allow the investigation of suppressive effects of antibodies/plasma from immune thrombocytopenia (ITP) patients. In this study, we developed a megakaryocyte culture with the utilization of human PB-HPCs in an efficient fashion resulting in the production of high purity megakaryocytes in a TPO-dependent manner.

The mononuclear fraction was collected from 180 mL of peripheral whole blood and CD34+ cells were isolated by a positive selection yielding the average of 5.5 x 105 ± 2.5 x 105 CD34+ cells (n = 18). Using 96-well tissue-culture plates and seeding 10,000 CD34+ cells/well, the average of 13 experiments in triplicate can be set up utilizing isolated CD34+ in an efficient manner. Capitalizing on a TPO dose-dependent megakaryocyte production experiment, 20 ng/mL was established as the TPO concentration which resulted in the production of mature megakaryocytes without reaching the plateau in megakaryopoiesis response. On day 11 of culture, the expression of megakaryocytic lineage (CD41/61+) and maturation (CD41/61+CD42+) markers peaked at 90.65% and 76.10%. In conclusion, this culture system has broad application for investigation of platelet disorders and drug discovery which can be accessible to all researchers.

abstract: Master of Science (MSc)


title: Female Impersonation and Patriarchal Resilience in Early Stuart England

contributor:
Gough, Melinda
Ostovich, Helen
Silcox, Mary
English and Cultural Studies

subject:
Early Modern Literature
early modern women
female impersonation
English Civil War
Sarah Jinner
Swetnam the Woman-Hater
Cultural History
Feminist, Gender, and Sexuality Studies
Literature in English, British Isles
Cultural History

abstract:
In seeking to explain why male authors assumed female pseudonyms in seventeenth-century literature, this dissertation explores male-to-female cross-dressing in Jacobean drama, effeminizing representations of parliament in Civil War propaganda, and parodies of women’s sexualized, political speech during the Interregnum and Restoration periods. My dissertation concludes that the sexualized female persona evolved over the course of the seventeenth century as a vehicle through which male authors could critique rival iterations of patriarchal hierarchy forwarded by Stuart kings and by parliament without challenging their own positions of masculine privilege within those hierarchies.

My first chapter explores the political critiques of Jacobean absolutism embedded in the cross-gender performance narratives of Ben Jonson’s Epicoene (1609) and the anonymous play Swetnam the Woman-Hater (1620). In my second chapter I link male-to-female drag’s ability to critique an absolutist patriarchal paradigm to the satirical attacks on parliamentary models of polyvocal patriarchal rule in 1640s print. My final chapter investigates how female authors often find themselves shut out of the political discussions that female impersonations spark by taking up Sarah Jinner’s almanacs of 1658-60. Jinner’s almanacs combine predictions of rampant sexual wantonness with a critique of the waning Protectorate regime. I examine how the pseudonymous response to those almanacs from “Sarah Ginnor” depoliticizes Jinner’s sexual commentary on the Protectorate government.

abstract: Doctor of Philosophy (PhD)


title: The Function of Pap in the Sinorhizobium meliloti Pap-Pit Low Affinity Phosphate Transport System

contributor:
Finan, Turlough M.
Elliot, Marie
Morton, Richard
Biology

subject:
S. meliloti
transporter
phosphate uptake
regulation
localization
Microbiology
Microbiology

abstract: Pap-Pit is a low affinity phosphate transporter found in S. meliloti and many other microorganisms. Pit is the transporter and Pap is the Pit accessory protein. Pap has been shown to be required for the function of Pap-Pit system in S. meliloti. In this study, pap-pit or pit alone from three species of bacteria have been expressed in trans in the E. coli Pi uptake mutants to check their ability to complement the Pi uptake deficiency of the hosts. A visualization tag, SNAP-tag, has been fused to S. meliloti Pap to help determine the subcellular localization of Pap. Here we show that there is an optimal level of Pap-Pit in the cells, and Pap appears to modulate this level to optimize the function of the system. We also demonstrate that Pap is probably localized intracellularly along the cell membrane. In addition, a S. meliloti pap-pit deletion strain has been prepared and to be used as the background strain for site-directed mutagenesis in Pap. The highly conserved surface amino acids in Pap have been identified to be the candidates for the site-directed mutagenesis.

abstract: Master of Science (MSc)