title: Dynamic Optimization, State Estimation and Control of Electric Arc Furnace Operation

contributor:
L.E. Swartz, Christopher
Chemical Engineering

abstract:
Electric arc furnaces (EAFs) are commonly used in the steel industry for production of steel by melting down the scrap metal and altering its chemistry. The highly energy intensive steelmaking operation is a complex batch process, and involves limited automation.
The main aim of this research is to develop a mathematical model and computational framework for on-line optimization-based decision support and control of EAF operation. A dynamic model of the EAF process is implemented and used within an optimization framework to determine the optimal input trajectories. Industrial implementation of the model-based optimization systems is envisaged to generate significant savings through reduced consumption of electric power, natural gas, oxygen, carbon and fluxes such as limestone and dolomite.
In the first part of the study, we present the advances made through this research toward implementing an economic optimization of EAF operations. The three key building blocks for a real-time implementation are discussed: a dynamic model, dynamic simulation and dynamic optimization. We also present the simulation and optimization deployments for the industrial members of the McMaster Steel Research Centre (SRC) through which users can interact with the model.
In the second part of the study, state estimation is investigated through implementing multi-rate Moving Horizon Estimation (MHE) for real-time model calibration.
A parameter estimation based multi-rate MHE framework is developed to handle measurements with different sampling rates. The MHE application is further extended for the full batch time with the use of a simultaneous solution approach. A novel MHE initialization scheme is also proposed to reduce the numerical computation time. The estimator showed strong performance in tracking the internal states in the presence of plant-model mismatch and measurement noise, improving from poor initial guesses of the states. The multi-rate MHE is then coupled with an economics-based dynamic optimizer to form an online decision support tool. %(DST). The DST is able to reconstruct the states and provide optimal decisions to operators in real-time despite the use of a nonlinear large-scale EAF model.
In the third part of the study, an energy management approach is introduced that effectively curtails the energy cost in real-time through the implementation of economically optimal operating decisions. An economics-oriented shrinking horizon nonlinear model predictive control (NMPC) algorithm that exploits time-varying electricity prices is coupled with a multi-rate MHE to form an integrated decision-making framework. A novel initialization scheme is also developed for obtaining fast on-line solutions of the economic NMPC and multi-rate MHE dynamic optimization problems. The energy usage optimization results indicated a significant reduction in the operating cost and peak electricity demand compared to the case where the electricity price profile is not updated.
In the fourth part of the study, a real-time dynamic optimization-based advisory system that employs a first-principles EAF model is introduced to support the operator decision making in real-time. Economically optimal process operation is achieved by employing the first-principles dynamic EAF model in the optimization formulation. A dynamic optimization calculation can be triggered by the operator at any point in the batch, an action that can be repeated multiple times during the batch.
The advisory system incorporates a multi-rate MHE that continually computes estimates of the process states utilizing current and past inputs and measurements.
End-point constraints and potential extension of the batch duration are handled through a multi-tiered optimization algorithm. Our case studies demonstrate a major economic improvement when the dynamic optimization-based advisory system is used.

abstract: Dissertation

abstract: Doctor of Philosophy (PhD)


title: Construction of a Herpes Simplex Virus Type 1 (HSV 1) Insertion Mutant Containing the Bacteriophage T4 Den V Gene: Genes that are Important for the UV Survival of HSV 1

title: Genes Important in the U. V. Survival of Herpes Simplex Virus

contributor:
Rainbow, Andrew
Biology

subject:
herpes
herpes simplex virus
gene
mutant
bacteriophage
uv

abstract: The den V gene from bacteriophage T4 codes for a small, pyrimidine dimer specific, endonuclease. Recent studies have shown that transfection of the gene into DNA excision repair deficient, Xeroderma Pigmentosum cells, can partially restore the excision repair ability of the cells and results in an increased resistance to UV light. In this study the den V gene has been inserted into Herpes Simplex Virus type 1 (HSV 1) in order to determine if HSV 1 can be used as a suitable vector for studying DNA repair genes. A 1.9 kb cartridge containing the den V gene, the 3' LTR of Rous Sarcoma Virus as the promoter, and the SV40 polyadenylation signals was inserted as the thymidine kinase locus of the virus. Properly initiated transcription form the construct, HDV 1, was verified by primer extension analysis. The Host cell reactivation of this virus and several other strains of HSV 1 were examined in normal and Xeroderma Pigmentosum cells. The results from these experiments suggest that both the viral DNA polymerase and thymidine kinase genes play important roles for the survival of UV irradiated HSV 1.

abstract: Thesis

abstract: Master of Science (MS)


title: Expression of Adenovirus Type-5 E1B Tumor Antigens in Escherichia Coli

title: Expression of Adenovirus Tumor Antigens in E. Coli

contributor:
Graham, F. L.
Biology

subject:
e. coli
tumor
antigens
adenovirus

abstract: The Ad5 E1B antigens of MW 58000 and 19000 are known to be involved in oncogenic transformation of mammalian cells. To obtain sufficient quantities of these proteins for biochemical studies on their mechanism of action, I have attempted to express the Ad5 E1B genes in Escherichia coli. Using the strategy developed by Guarente et al. (1980), I have constructed plasmids which have the trancriptional and translational controls of the E. coli lac operon linked 5' of the 19k and 58k coding sequences. One plasmid was shown to synthesize high levels of a stable, immunoreactive 19k analogue consisting of 19k with 29 adenovirus-coded ribosome-binding sequence is functional in directing translation of this protein. Synthesis of 58k was not demonstrated, perhaps the result of [protein instability in E. coli. However, immunoreactive proteins which may correspond to the amino terminal region of 58k were demonstrated.

abstract: Thesis

abstract: Master of Science (MS)


title: Identification of Phosphorylation Sites at the Carboxy Terminus of the 55-K (496R) Adenovirus Type 5 E1B Protein

title: Phosphorylation of the AD5 E1B 55K Protein

contributor:
Branton, P. E.
Graham, F. L.
Biology

subject: phosphorylation carboxy terminus adenovirus protein

abstract: The 55K product of early region 1B (E1B) of human adenoviruses is required for viral replication and participates in cell transformation. Both biochemical and genetic approaches have been used to show that this 496-residue (496R) protein of adenovirus (Ad5) is phosphorylated at both serine and threonine residues at sites near the carboxy terminus within sequences characteristic of a casein kinase II. Mutations which converted serines 490 and 491 to alanine residues decreased virus replication and reduced the efficiency of transformation of primary baby rat kidney cells, suggesting that phosphorylation at these sites is of importance in the regulation of 55K biological activity.

abstract: Thesis

abstract: Master of Science (MS)


title: Physician Utilization in Quebec 1987 and 1992-93; the Impact of Regionalization in a Cost-Constrained Climate

contributor:
Eyles, John
Geography

subject: quebec physician

abstract: The objective of this thesis was to determine whether the development of a regionalized health care system in Quebec has enabled this province to meet the health services needs of its citizens in the face of Federal cuts to health care. This was accomplished through the quantitative analysis of the relationship between the incidence of family physician use and self-assessed health status (a proxy for need for care) in Quebec. The data for this study came from the 1987 and 1992-93 Santé Quebec which is a weighted random sample of the citizens living in Quebec aged 15 years and over. Using Aday and Andersen's (1974) framework for the study of access to medical care, the relationship between physician utilization and need, predisposing and enabling characteristics of individuals were analyzed for both years of the survey. The results at the provincial level revealed that need was the most important determinant of use and further that those with higher levels of need had a greater probability of use. Yet, non-need variables were also important in determining use. Specifically, the importance of predisposing factors increased from 1987 to 1992-93. The data were also partitioned by need level and by health region (15 of the 18 health regions in Quebec were included in the Santé Quebec survey). These results showed that need level was important in influencing the associations between the enabling and predisposing variables and utilization. When the data were disaggregated to the regional level, a complex pattern of utilization behaviour appeared which was not apparent at the provincial level. The results of this analysis show that while regionalization has allowed the province of Quebec to better identify the health care needs of Quebec residents, variations in utilization still exist. It has also shown that while socio-economic barriers to use have diminished, the importance of socio-demographic characteristics have increased over time. There are three important conclusions which can be drawn from this analysis; 1) need is an important determinant of physician use, 2) temporal and spatial data analysis should be included in investigations of this type as they can reveal important variations which would otherwise be unnoticed, and 3) further research in this area should incorporate the use of both qualitative and quantitative analysis in order to better identify the individual processes involved in using physician services.

abstract: Thesis

abstract: Master of Arts (MA)


title: Identification of the Adenovirus Type 12 Gene Product(s) Required for Induction of Chromosomal Aberrations in Human Cells

contributor:
Bacchetti, Silvia
Biology

subject:
adenovirus
gene
induction
chromosome
human cell
aberration

abstract:
Unlike most RNA and DNA containing viruses, which induce cytogenetic damage at random sites throughout the human genome, the highly oncogenic adenovirus type 12 is also capable of inducing damage at specific chromosomal sites. Infection of human embryonic retinal or kidney cells with Ad12 results in the induction of heterochromatic gaps at specific (17q21-22, 1p36, 1q21, and 1q42-43) and random sites in the cellular chromosome. Previous work by Durnam et al. (1986) demonstrated that the viral early region 1 (E1) is sufficient for the induction of damage at band 17q21-22. The objective of the present study was to 1) identify the Ad12 E1 gene product(s) required for the induction of aberrations in human diploid cells, and 2) to determine whether the same or different functions are involved in the induction of damage at specific and random sites. To this end, adenovirus type 12/adenovirus type 5 recombinants with hybrid E1 sequences as well as viruses with mutations in the Ad12 E1B genes were used to map the Ad12 E1 function(s) required for the induction of chromosomal aberrations. The results of this study indicate that the expression of E1A proteins is not sufficient for this
effect. On the other hand, mutations within the E1B 55Kd protein but not the E1B 19Kd protein were found to affect the ability of the virus to induce both specific and random damage (Schramayr et al., 1990).

abstract: Thesis

abstract: Master of Science (MS)


title: Sequences in Adenovirus 5 E1A Gene that are Required for Transcriptional Activation, Enhancer Repression, and Oncogenic Transformation

title: Functional Domains in Adenovirus 5 E1A

contributor:
Bayley, S. T.
Biochemistry

subject: adenovirus gene transcription repression oncogenic transform enhance activate

abstract: The E1A gene of adenovirus 5 carries out a number of functions in infection and oncogenic transformation, including the transcriptional activation of viral and cellular genes, the repression of transcriptional enhancers, and cooperation with the adenovirus E1B gene or with the ras oncegene to transform primary cells. The purpose of this work was to investigate the mechanism of action of E1A, by determining the regions of the proteins that are required for these functions. Deletion and point mutations were made in the region unique to the larger E1A mRNA, by exonuclease digestion and deletion loop mutagenesis respectively. These mutants and a series of mutants which delete sequences spanning the entire coding region, were examined for their effect on transcriptional activation, enhancer repression, and transformation. The region which, when deleted, rendered E1A defective for transcriptional activation was found to be confined to the region unique to the 13s mRNA and the beginning of exon 2. Mutations in three regions, all within the 12s exon 1, affected repression activity. The first two, the N terminal region of the protein, and a region, CR1, conserved between adenovirus serotypes, were essential for repression activity. The third region, at the end of exon 1 of the 12s mRNA, was probably only indirectly involved in repression. Deletions in three regions of exon 1 resulted in a loss of the transforming function of E1A. The first two corresponded to the regions required for repression, suggesting that enhancer repression is a component of transformation. The third region, containing CR2, also conserved between adenovirus serotypes, is functionally distinct from the other two and appeared to affect the morphology of the transformants. These two functions did not operate efficiently when present ion separate plasmids. The 13s unique region and exon 2 were not required for transformation but the loss of the transactivation function of E1A did result in an increased adhesiveness of the transformants.

abstract: Thesis

abstract: Doctor of Philosophy (PhD)


title: Northern Blot Hybridizations of RNA from AD 5 Transformed Cell Lines

contributor:
Bayley, S. T.
Biology

subject: blot hybrid cell RNA

abstract: Researchers have tried to identify the viral factor involved in making a transformed cell oncogenic in newborn hamsters. Although studies on the viral proteins from transformed cells failed to show a relationship to oncogenicity, this study intended to identify and map viral mRNAs from these transformed cell lines in order to test the same relationship. Northern blot hybridizations were used to study the AD5 E1 region specific mRNAs from ten transformed cell lines. These cell lines had been transformed with either virus, total restriction endonuclease digested viral DNA, or specific fragments of viral DNA from the left hand end of the genome. All of these lines were selected for their varying oncogenicity in newborn hamsters, and for the size of the transforming fragment. Ad5 DNA fragments inserted into recombinant plasmids were used as the probes for detection of mRNAs, providing tools for mapping transformed cell mRNAs to specific regions of the Ad5 genome. The results failed to show a relationship between E1 region mRNA production and oncogenicity, but did reveal unusual mRNA transcription patterns from most of the cell lines. All the transformed cell lines tested appeared to have only an E1B 22s mRNA as the major mRNA from that region. An E1A/E1B cotranscript was identified as well as viral/cell chimeric mRNAs. These chimeras were due to either run-off transcription or to preinitiation in cellular sequences.

abstract: Thesis

abstract: Master of Science (MS)


title: Analysis of Adenovirus Type 5 Early Region 1 A Insertion Mutants

contributor:
Graham, Frank
Biology

subject: adenovirus mutant insertion

abstract: Adenovirus Early region 1A(E1A) plays an important role in viral lytic infection and serves as a useful tool in the study of molecular mechanisms of gene regulation and gene expression. In addition to the ability of E1A functions to induce transactivation and transrepression of gene expression, E1A products play a critical role in transformation by adenovirus. A technique employing linker insertion mutagenesis scanning most of the E1A major regions was developed in this laboratory to systematically study the effect of E1A mutations on transcriptional transactivation and trans-repression functions as well as transforming ability (Bautista 1989). Results obtained showed that the unique region was the region primarily for transactivation in agreement with a wealth of other data and that transrepression was sensitive to insertions in CRII (conserved region II) and a region immediately following the unique region at the beginning of Exon II. However, transformation was not affected by repression-sensitive mutants. This thesis describes further studies carried out to create additional insertion mutants within conserved region of E1A to confirm and extend the results obtained by Bautista. A synthetic oligonucleotide of 39 base pair (13 aa) was introduced into two additional restriction sites in this region. As in previous work, the oligonucleotide could be inserted in either of two possible orientations. In one orientation, all three reading frames were open whereas all three reading frames of the other orientation contained stop codons which resulted in truncation of the E1A protein at the site of insertion. In addition, the insertion oligomer was designed with flanking BamHI sites to provide the opportunity of collapsing the full length insertion to a 6 base pair (2 aa) insertion. This allowed comparison to be made between mutations containing 13 aa insertions and those with 2 aa inserts at the same site to see how different oligopeptide segments would influence different functions. Trans-repression assays utilized a novel β-galactosidase assay developed by Bautista for characterizing CRII mutants expressing only the 12S product. The results suggested that CRII was an important region in terms of ability of E1A to transrepress. This negative regulatory function was sensitive to full length 13 aa insertions but not 2 aa inserts for those mutants made to date. Transforming ability of all CRII mutants was more or less impaired. Transactivation activity was not reduced by insertion at CRII except for mutants which were in the reverse orientation and had stop codons in their reading frame thus terminating the translation upstream of the unique region. In an effort to identify functional temperature sensitive mutants, DNA-mediated transformation assays of mutants within E1A functional regions were carried out at different temperatures. Two transformation temperature sensitive mutants were identified which failed to produce transformed colonies at 38.5°C but transformed efficiently at 32°C. No replication temperature sensitive mutants were identified among 61 mutant viruses.

abstract: Thesis

abstract: Master of Science (MS)


title: Construction and Characterization of an Adenovirus Vector Containing a Bicistronic IL-12 Expression Cassette

subject: adenovirus bicistronic cassette construction

abstract: Gene therapy, although a young field, has become an intense area of study for the potential treatment of many inborn or acquired diseases, including cancer. The systemic or intratumoural delivery of cytokines and other immune system modulators has shown that protective, specific immunity to tumours is possible and effective. Adenovirus is an extremely useful vehicle for the transfer of genes, due to the ease of its preparation, its ability to be grown to high concentrations, and its capacity to infect a wide variety of cell types, including nonproliferating cells. Although a great deal of research has been done, there are a number of possible improvements, specifically, the consistency and range of transgene expression. The HCMV IE promoter/enhancer, used in a wide range of vector systems, has proven to drive lower levels of transgene expression in murine cells as compared to human cells. The use of the MCMV IE promoter/enhancer within the context of an adenovirus vector system has been investigated to see if more consistent transgene expression is possible in murine models. Studies with a luciferase reporter gene have demonstrated that in all cell types, the MCMV IE promoter/enhancer drives transgene expression to equivalent or higher levels than the HCMV IE promoter/enhancer. Our lab has investigated adenovirus as an intrtumoural gene delivery vehicle for various cytokines, including IL-12. However, the current IL-12-expressing vector carries the IL-12p35 subunit in E1 and the IL-12p40 subunit in E3. A homologous recombination event between E1 sequences in the viral genome and those that are integrated into 293 cells, would produce a replication-competent adenovirus expressing high levels of the p40 subunit, which is a potential antagonist to IL-12. A bicistronic adenovirus vector using the poliovirus IRES was constructed containing all IL-12 coding sequences in E1 to avoid this potential hazard. This virus expressed between 60 ng to 2.5 μg of IL-12/10⁶ cells, depending on the cell type. These levels of expression, however are 7-30-fold lower than those driven by previous IL-12 vector. Although this new virus is not appropriate for tumour immunotherapy, the shuttle plasmids developed during its construction have many potential uses.

abstract: Thesis

abstract: Master of Science (MS)


title: Effects of Inhibition of Protein Synthesis on Adenovirus 5 Early Gene Expression

title: Studies on AD5 Early Gene Expression

subject: protein adenovirus gene expression inhibition

abstract: Previous studies have shown that a functional Ad 5 E1A protein from the 13 S mRNA is required during lytic infection for activation of early viral gene expression (Jones and Shenk, 1979b; Berk et al, 1979; Montell et al, 1982). The mechanism by which E1A exerts this regulatory function is still unclear and has been the subject of intense investigation (Persson et al, 1981a; Katze et al, 1981, 1983; Nevins, 1981; Shaw and Ziff, 1982). Most of these recent investigations have relied on the use of metabolic inhibitors such as cycloheximide to eliminate protein synthesis as a means of determining the role of viral and host proteins in the regulatory process. The results from these studies have been inconsistent. The purpose of this research project has been to re-examine the regulation of Ad 5 early gene expression without the use of drug inhibitors. In this study tsH1 cells, a mutant CHO cell line which at temperatures above 37°C are inhibited in protein synthesis, were used. At critical times during the course of wild type or host-range 1 infection of tsH1 cells, protein synthesis was inhibited and Early Region 4 expression was examined. In every case, efficient E4 expression was dependent on functional E1A protein and this requirement could not be replaced by simply inhibiting protein synthesis. The results are discussed in relation to models proposed to explain the regulation of Ad 5 Early Gene Expression.

abstract: Thesis

abstract: Master of Science (MS)


title: An Evaluation of a Thin Film X-Ray Imaging Sensor with Laser Read-Out

contributor:
Ramanathan, N.
Engineering Physics

subject: x-ray film sensor laser imaging

abstract: This report describes the industrial research work done during the summer of 1986. The main objective of this research was to develop a high resolution X-ray imaging system for mammographic applications. The basic set up of such system is quite simple: it consists of an x-ray tube; an x-ray sensor (which is coupled to the read out electronics), and the optical scanner assembly which serves as the reading device. Based on the feasibility studies carried out two years ago, much of the work during the past summer was targeted to improve the existing prototype, and to test out the new x-ray sensors. A new optical assembly was built for this system, which was used to examine the optical resolution of the new sensors. Experimental results were analysed using calculations of the resolution (MTF) of the sensors, and the results indicate that the latest sensors are capable of resolving a 1mm diameter object. Unfortunately, due to the limitation of time, no x-ray exposure test were done on test targets. However, a theoretical model describing the x-ray sensors responses to x-ray radiation and laser light (based on previous work carried out by A. Zermeno et al, 1979) is presented here. Also, the results of the research work, carried out since the feasibility study up until the end of the summer of 1986, indicate that the sensors have a great potential for achieving limiting resolutions in the range of "0.1mm" objects.

abstract: Thesis

abstract: Master of Engineering (ME)


title: OPTIMAL CLOSURES IN HYDRODYNAMIC MODELS

contributor:
Protas, Bartosz
Mathematics

subject:
Mathematics
Closure models
Kuramoto-Sivashinsky
Smagorinsky model
Large-Eddy Simulation
Optimization

abstract: In this work, we investigate the performance limitations characterizing certain common closure models for nonlinear models of fluid flow. The need for closures arises when for computational reasons first-principles models, such as the Navier-Stokes equations, are replaced with their simplified (filtered) versions such as the Large-Eddy Simulation (LES). In the present work, we focus on a simple model problem based on the 1D Kuramoto-Sivashinsky equation with a Smagorinsky-type eddy-viscosity closure model. The eddy viscosity is assumed to be a function of the state (flow) variable whose optimal functional form is determined in a very general form in the continuous setting. It is found by solving a PDE-constrained optimization problem in which the least-squares error between the output of the LES and the true flow evolution is minimized with respect to the functional form of the eddy viscosity. This problem is solved using a gradient-based technique utilizing a suitable adjoint-based variational data-assimilation approach implemented in the optimize-then-discretize setting using state-of-the-art techniques. The numerical computations are thoroughly validated. The obtained results indicate how the standard Smagorinsky closure model can be refined such that the corresponding LES evolution approximates more accurately the evolution of the original (unfiltered) flow.

abstract: Thesis

abstract: Master of Science (MSc)


title: Design and Testing of a Noninvasive Steady-State Device for the Measurement of Optical Properties of Tissues in the 900-1400nm Wavelength Region

title: Device for Measurement of Optical Properties of Tissues: 900-1400nm

contributor:
Patterson, Michael
Physics

subject:
optic
wavelength
non invasive
steady
tissue
measure

abstract:
Thesis
Master of Science (MS)


title: Alternative Summary Indices: PLC and ASC for the Summary Receiver Operating Charcteristic (SROC) Curve

contributor:
Walter, Stephen
Statistics

subject:
ROC curve
SROC curve
meta-analysis
projected length of the curve
area swept out by the curve
homogeneous

abstract:
Thesis
Master of Science (MS)


title: Construction and Characterization of Human Adenovirus Recombinants Expressing the Vesicular Stomatitis Virus Glycoprotein Gene

contributor:
Prevec, Ludvik
Biology

subject:
adenovirus
gene
glycoprotein
stomatitis
protein
vesicular stomatitis

abstract: The potential of human adenovirus (Ad) to serve as a vector for expression of heterologous genes was evaluated. An experimental gene, consisting of sequences coding for the glycoprotein of vesicular stomatitis virus (VSV) attached to the promoter and polyadenylation signal of the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene, was inserted into early region 3 of adenovirus, in both orientations. The TK promoter was functional in both orientations. The TK promoter was functional in both orientations and responded to trans-activation by HSV infection. Abundant expression of VSV G however depended on the presence of a second transcript. This transcript was present only in the recombinant carrying the insertion in the orientation parallel to the E3 promoter (AdG12) and was initiated upstream of the insertion, within Ad sequences. The potential of Ad recombinants to serve as vaccine vectors was investigated using the recombinant AdG12. Antibody against VSV G was induced in cows, pigs, and dogs in response to infection with AdG12. Protection of mice, immunized with AdG12, against a lethal challenge with VSV demonstrated the biological effectiveness of this immune response.

abstract: Thesis

abstract: Master of Science (MS)


title: Transformation and Plaque Forming Ability of Adenovirus Type 5 E1A Insertion Mutants

subject: E1A adenovirus mutant plaque

abstract: The E1 region of the group C adenoviruses is able to induce oncogenic transformation of rodent cells in culture and is also necessary for the efficient transcription of other viral genes. The proteins of the E1a transcription unit have been shown to play a pivotal role in both of these activities. In order to better understand the regions of the E1a proteins required for transformation and viral growth a program of random insertion mutagenesis was undertaken by D. S. Bautista to help identify important domains. The 39bp linker insertion oligonucleotides were designed to encode a 13 amino acid in frame insertion in one orientation or a closed reading frame insertion in the opposite orientation. As well the insertion mutants could be collapsed by digestion with BamHI to generate a 2 amino acid in frame insertion. Using this method all three types of mutants were generated at 18 different sites within the E1a coding sequences. The purpose of this project was to assay these E1a mutants for the ability to cooperate with EJb in the transformation of primary baby rat kidney cells using DNA-mediated transfection and also to 'rescue' the mutants into infectious virus and study the ability of the mutant virus to replicate on HeLa cells. Results showed that only closed reading frame mutations upstream of the unique region were completely negative for transformation. Conversely, 13aa or 2aa insertions outside of the unique region impaired but did not abolish transformation. However 8 of the 9 insertions in the unique region of the 289R protein of E1a were defective for transformation of BRK cells in E1a plus E1b DNA-mediated transformation assays. To determine whether the unique region played a direct role in the transformation process or if it had an indirect role such as the transactivation of E1b the transformation assay was carried out using selective media that allow~ growth of foci transformed by E1a alone. Results from this assay showed that the unique region mutants combined with E1b were able to transform with about the same efficiency as E1a alone. The transformation assay was also performed using the unique region mutants in an E1a only background cotransfected with the EJ-ras oncogene. Results from these experiments showed that the unique region mutants in an E1a only background could cooperate with ras in transformation as well as wild-type E1a. From these results it was concluded that the unique region does not play a direct role in transformation by E1 but is required for the efficient expression of E1b which results in wild type transforming frequencies. The actual role of E1b in transformation is unknown. The insertion mutants were also 'rescued' back into infectious virus to study their effect on the ability of the viruses to replicate. The results showed that only viruses in which the unique region was either eliminated or altered were defective for growth on HeLa cells. Transactivation assays carried out by D. Bautista showed results which were comparable to results of infectivity assays. Taken together the results suggest that only the unique region is required for transactivation and only the ability of E1a to transactivate is of importance for viral replication in HeLa cells.

abstract: Thesis

abstract: Master of Science (MS)


title: Genetic and Viral Specificity of the Cell-Mediated Immune Response to Pichinde Virus in Mice

contributor:
Rosenthal, K. L.
Biology

subject: genetic virus cell mice immune pichinde

abstract: This study was undertaken to examine the genetic and viral target specificity of the cell-mediated immune response to Pichinde virus (PV) in mice, to ultimately facilitate the cloning of anti-PV cytotoxic T lymphocytes (CTL). Cloned populations provide a system for the study of the generation, function, and mechanism of action of CTL in viral infections. CTL are often genetically restricted to either the K or D end of H-2 Class I. Cytotoxicity assays performed using primary immunized murine splenocytes showed predominant restriction to the K locus in H-2ᵇ C57B1/6 mice. In limiting dilution assays, a significant population of anti-PV CTL (approximately one-half) were regulated by H-2Kᵇ. A panel of temperature sensitive (ts) mutants of wild type (wt) PV was used to study the viral target specificity of anti-PV CTL. These mutants were grouped according to surface expression and internal production of viral molecules as detected by indirect immunofluorescence and polyacrylamide gel electrophoresis respectively. Cytotoxicity assays with target cells infected with these mutants provided equivocal results regarding whether the major target antigen was an internally or externally expressed viral protein. The results suggest that surface expression of viral proteins may be related to target specificity in the anti-PV cytotoxic response. One temperature sensitive mutant, ts 488, provided a superior target for H-2ᵇ effectors than wt PV when used to infect 5R (H-2Kᵇ compatible) target cells. Limiting dilution studies indicated that anti-ts 488 CTL were predominantly regulated by H-2Kᵇ: approximately three-quarters of the CTL precursors were restricted to that end. Furthermore, limiting dilution assays indicated that ts 488 and wt PV were not fully cross reactive in generating lytic effectors. As a result, the mutant might not facilitate the generation of cloned lines of anti-wt PV CTL. The mutant and its augmented regulation by H-2Kᵇ may provide a novel system for the examination of the association and recognition of viral plus "self" antigens at the level of antigen presentation and target identification.

abstract: Thesis

abstract: Master of Science (MS)


title: Macromolecule Transport in Tumours: Mathematical Modelling and Experimental Studies

title: Macromolecule Transport in Tumours

contributor:
Kwok, C. S.
Physics

subject: macromolecule molecule tumour mathematics experiment

abstract: The delivery of immunoreactive macromolecules to tumour cells in solid, heterogeneously perfused tumours is a major problem in the effectiveness of immunotherapy. To help optimize the new experimental treatment method, a published mathematical model of macromolecule transport (Baxter & Jain 1989,1990,199la) was appraised and verified experimentally. Computational and analytical tools were developed to predict the interstitial plasma fluid pressure and velocity distributions in well perfused spherical tumours. Their published analytical solutions of the formulation were found to have some errors and were corrected in this work. To check the validity of the formulation, a series of animal experiments was performed to quantify the total vascular volume, and plasma fluid extravasation rate in SKOV3ipl human ovarian tumour xenografts in nude mice. The results compared well with the theoretically predicted total plasma fluid extravasation rate. Computer codes were also developed to predict the spatial and temporal distributions of intact IgG and its F(ab')₂ and Fab/Fab' fragments in well perfused spherical tumours using the formulation proposed by Baxter & Jain (1989,1990,1991a). The cases of non-binding and binding macromolecules were treated separately. The codes for both the interstitial pressure and macromolecule distributions were written to include a radially variable vessel surface area for transcapillary exchange per unit volume of tumour tissue (SN). The sensitivity of the overall tumour perfusion to variation of (a) the macromolecule m.w., binding affinity, and metabolism, (b) SN, tumour radius, and (c) microvascular permeability were investigated. Comparison of the theoretical predictions with available experimental data leads to the realization of a number of shortcomings in the previously proposed formulations. Finally, a computational method for deriving the effective spherically symmetric spatial distributions for the vascular volume density, and SN from tumour serial sections was developed. This bridges the gap between the actual topology of vascular distributions in tumours and the format of current formulations.

abstract: Thesis

abstract: Master of Science (MS)


title: The Ability of Human Adenovirus Type 5 to Replicate in MDBK, ADCK, HELA & L Cells

title: The Replication of hAd5 in MDBK, MDCK, HELA & L Cells

subject: adenovirus human host replicate

abstract:
Thesis
Master of Science (MS)


title: Design and Control of an Isolated Battery-Driven Grid Interface with Three-Phase Dual-Active-Bridge Converter

contributor:
Emadi, Ali
Electrical and Computer Engineering

subject:
Power electronics
Energy storage system
Isolated DC/DC converter
Single phase inverter
Hybrid capacitor bank
Second harmonic current

abstract:
Battery energy storage system (BESS) is promising to be implemented in residential applications for supporting PV integration, load shifting, and backup power purposes. For this application, 48V second-life battery draws more and more attentions for their cost-effectiveness, safe voltage level, reliability, and potential large market. This thesis proposes the comprehensive control and design of an isolated battery-driven grid interface (IBDGI) with the dual-active-bridge (DAB) converter for residential applications with 48V battery pack.

The three-phase DAB converter is a promising candidate as the front-end DC/DC converter in the two-stage IBDGI due to its high efficiency, high power density, and low capacitance requirement. An effective design strategy for the three-phase DAB converter is proposed based on the zero-voltage-switching (ZVS) zone and back-ow power to achieve high efficiency for a wide operating voltage range and different load conditions. Based on the power loss model, an easily-implemented variable switching frequency operating method is proposed to further increase the efficiency at light load conditions.

The dead-time effect is observed in the three-phase DAB converter. To avoid the dead-time effect and better understand the phenomena, a comprehensive analysis is proposed. All the cases of the dead-time effect in the three-phase DAB converter are analyzed in terms of the buck, boost, and matching states. The expressions of the transmission power, constraint conditions, and key time of the dead-time effect are derived for each state. The operation waveforms of the dead-time effect are also presented.
The hybrid capacitor bank composed by the LC resonant  lter with electrolytic
capacitor and  lm capacitor is utilized for the DC bus of the IBGDI. The electrolytic
capacitors work as passive decoupling purpose while the  lm capacitor is responsible
for high switching harmonic  ltering. Moreover, a current sharing method between
the hybrid capacitor bank is proposed to extend the electrolytic capacitor's life.
The LCL single-phase inverter is applied for the downstream of the IBDGI. A
step-by-step design procedure of the LCL  lter with passive damping is proposed for
the 120V/240V dual grid-tied and standalone modes. The PR controllers are also
designed for the LCL inverter for standalone and grid-tied modes.
At the system level, a novel second harmonic current (SHC) reduction strategy is
proposed for the IBDGI with the three-phase DAB converter by adding a load current
feedforward (LCFF) path to the DAB voltage closed-loop controller. This method will
suppress the SHC without modi cations of the original controller's bandwidth, which
make it easy to be implemented. The small-signal model of the three-phase DAB
converter is provided and veri ed by the step response. The parameter sensitivity
analysis for the LCFF method is proposed to show that the SHC is well suppressed
within ±20% parameter error.
The proposed converter and control methods are veri ed by simulation and experimental
results.

abstract: Thesis

abstract: Doctor of Philosophy (PhD)


title: Development of High-throughput Membrane Filtration Techniques for Biological and Environmental Applications

contributor:
Latulippe, David
Chemical Engineering

subject: Membrane filtration

subject: Ultrafiltration

subject: Downstream bio-processing

subject: High-throughput (HT) testing

subject: Wastewater treatment

subject: Hollow-fiber membranes

subject: Humic acids

subject: High-throughput filtration

subject: Design-of-experiments (DOE)

subject: Process optimization

subject: Microscale filtration

subject: Microfluidic flow control system

subject: Stirred well filtration

subject: SWF

subject: High-throughput hollow-fiber module

subject: HT-HF

subject: Constant TMP

subject: Constant flux

subject: Multi-modal filtration

subject: Bioseparation

subject: MMFC

subject: Microscale parallel-structured, cross-flow filtration

subject: MS-PS-CFF

subject: PEG

subject: Dextran

subject: FITC-Dextran

subject: BSA

subject: DNA

subject: IgG

subject: α-lactalbumin

subject: Biomolecule separation

subject: Module hydrodynamics

subject: Concentration polarization

subject: Membrane fouling

subject: Micromixing

subject: Omega™ membrane

subject: Microscale processing

subject: Fouling test

subject: PVDF membrane

subject: Surface modification

subject: Polydopamine

subject: Membrane cleaning

subject: Membrane backwashing

subject: Sodium alginate

subject: Polyethersulfone

subject: PES

subject: Hydraulic permeability

subject: Membrane permeability

subject: ZeeWeed® membrane

subject: Filtration ionic strength

subject: Filtration pH

subject: Solution conditions

subject: Water treatment

subject: Environmental separations

subject: Biological separations

abstract: Membrane filtration processes are widely utilized across different industrial sectors for biological and environmental separations. Examples of the former are sterile filtration and protein fractionation via microfiltration (MF) and ultrafiltration (UF) while drinking water treatment, tertiary treatment of wastewater, water reuse and desalination via MF, UF, nanofiltration (NF) and reverse-osmosis (RO) are examples of the latter. A common misconception is that the performance of membrane separation is solely dependent on the membrane pore size, whereas a multitude of parameters including solution conditions, solute concentration, presence of specific ions, hydrodynamic conditions, membrane structure and surface properties can significantly influence the separation performance and the membrane’s fouling propensity. The conventional approach for studying filtration performance is to use a single lab- or pilot-scale module and perform numerous experiments in a sequential manner which is both time-consuming and requires large amounts of material. Alternatively, high-throughput (HT) techniques, defined as the miniaturized version of conventional unit operations which allow for multiple experiments to be run in parallel and require a small amount of sample, can be employed. There is a growing interest in the use of HT techniques to speed up the testing and optimization of membrane-based separations. In this work, different HT screening approaches are developed and utilized for the evaluation and optimization of filtration performance using flat-sheet and hollow-fiber (HF) membranes used in biological and environmental separations. The effects of various process factors were evaluated on the separation of different biomolecules by combining a HT filtration method using flat-sheet UF membranes and design-of-experiments methods. Additionally, a novel HT platform was introduced for multi-modal (constant transmembrane pressure vs. constant flux) testing of flat-sheet membranes used in bio-separations. Furthermore, the first-ever HT modules for parallel testing of HF membranes were developed for rapid fouling tests as well as extended filtration evaluation experiments. The usefulness of the modules was demonstrated by evaluating the filtration performance of different foulants under various operating conditions as well as running surface modification experiments. The techniques described herein can be employed for rapid determination of the optimal combination of conditions that result in the best filtration performance for different membrane separation applications and thus eliminate the need to perform numerous conventional lab-scale tests. Overall, more than 250 filtration tests and 350 hydraulic permeability measurements were performed and analyzed using the HT platforms developed in this thesis.

abstract: Thesis

abstract: Doctor of Philosophy (PhD)

abstract: Membrane filtration is widely used as a key separation process in different industries. For example, microfiltration (MF) and ultrafiltration (UF) are used for sterilization and purification of bio-products. Furthermore, MF, UF and reverse-osmosis (RO) are used for drinking water and wastewater treatment. A common misconception is that membrane filtration is a process solely based on the pore size of the membrane whereas numerous factors can significantly affect the performance. Conventionally, a large number of lab- or full-scale experiments are performed to find the optimum operating conditions for each filtration process. High-throughput (HT) techniques are powerful methods to accelerate the pace of process optimization—they allow for multiple experiments to be run in parallel and require smaller amounts of sample. This thesis focuses on the development of different HT techniques that require a minimal amount of sample for parallel testing and optimization of membrane filtration processes with applications in environmental and biological separations. The introduced techniques can reduce the amount of sample used in each test between 10-50 times and accelerate process development and optimization by running parallel tests.


title: Investigating the Role of Dectin-1 as a Marker of Profibrotic Macrophages in the Progression of Pulmonary Fibrosis

title: Alternatively activated macrophage markers and idiopathic pulmonary fibrosis

contributor:
Ask, Kjetil
Medical Sciences

subject:
interstitial lung diseases
macrophages
alternatively activated macrophages
idiopathic pulmonary fibrosis
dectin-1
clec7a
fibrosis

abstract: An estimated 45% of all deaths can be attributed to various chronic fibroproliferative diseases. Idiopathic pulmonary fibrosis (IPF) is the most common form of interstitial lung disease which is characterized by progressive decline in lung function. While the pathogenesis of IPF is not fully understood, alternatively activated macrophages (M2) have been implicated as a key contributor to the fibrotic process. The plasticity of macrophages in vivo challenges the ability to specifically target the M2 macrophage phenotype across species. Previous bioinformatic analysis from our lab identified Dectin-1/Clec7a as a unique marker of M2 macrophages in both human and murine model systems. The expression of the transmembrane receptor Dectin-1 has not been elucidated in the context of pulmonary fibrosis. To prevent the progression of fibrosis by targeting alternatively activated macrophages, we investigated the expression of Dectin-1 in IPF and an experimental model of fibrotic lung disease. Our data demonstrated that while protein expression of Dectin-1 was increased in archived lung tissues of patients with IPF, mRNA expression of this receptor was downregulated in the tissues of these IPF patients. Gene expression of Dectin-1 was shown to be increased in monocyte-derived macrophages, further suggesting a circulatory component contributing to lung fibrosis. As expected, we confirmed that Dectin-1 was highly expressed past the injury phase of the bleomycin-model of induced pulmonary fibrosis which aligns with the increased immune infiltrates at this time point. Preliminary work into the time dependency of the resolution phase of the bleomycin-induced model of lung fibrosis was shown. All in all, our data suggests that Dectin-1 may be a useful marker in characterizing and differentiating phenotypes of macrophages implicated in the fibrotic process. Future efforts aim to gain insight into the functional requirement of Dectin-1 in the alternative activation of profibrotic macrophages to identify novel therapeutic targets for fibrotic lung disease.

abstract: Thesis

abstract: Master of Science (MSc)


title: A Study on the Functions of the E1B Minor Products of Adenovirus Type 5

contributor:
Branton, P. E.
Biology

subject: adenovirus function minor product

abstract: The E1b transforming region of Adenovirus type 5 encodes minor products of 93R and 156R in addition to the more abundant proteins 178R, 496R, and 84R. The goal of this study was to elucidate the function of 93R and 156R to gain a better understanding of their role in oncogenic transformation and productive infection. Mutant viruses were constructed, whose normal splicing pattern was disrupted by point mutations in the 3' acceptor sites for the 1.26 and 1.31Kb mRNAs, which code for the 156R and 93R products, respectively. In the construction of these mutations, it was necessary to ensure that they did not affect the coding region for 496R. These mutants produced transformed foci in primary rat kidney cells with wild type efficiencies in DNA-mediated transformation assays. In the mutant designed to eliminate 156R, although the two wild type 156R species were absent, two new species running slightly faster on SDS-PAGE were detected. These proteins were recognized by sera specific to both the N- and C-termini of 496R, suggesting there utilization of an in-frame cryptic splice acceptor site. Use of this site probably resulted in the production of a mRNA encoding a modified 156R. These mutant proteins also seemed to be produced at the expense of 496R. The mutant designed to eliminate 93R grew with titres equivalent to wild type dl309, yet it was not clear whether a modified protein was produced in this case as well.

abstract: Thesis

abstract: Master of Science (MS)


title: Examining the Role of P53 in Radiation-Induced Mutations at ESTR Loci

title: The Role of P53 in Radiation-Induced ESTR Mutations

contributor:
Quinn, James
Biology

subject: p53 radiation mutation loci ESTR

abstract: It is well known that ionizing radiation is genotoxic, and can trigger heritable mutations in the germ cells of an animal. Recently, researchers have used hypervariable expanded simple tandem repeat (ESTR) regions of DNA to explore this phenomenon. ESTRs facilitate the examination of induced genetic mutations using relatively low radiation doses and fewer mice than more traditional approaches. Numerous studies have examined the responses of ESTRs to radiation in the germ line; however the mechanism behind germ line mutations at ESTR loci is poorly understood. Current hypotheses propose that error-prone DNA repair, which allows for misalignment of DNA strands through replication slippage produces in changes in ESTR size. P53 is involved in DNA replication as well as repair of DNA damage, apoptosis and other cancer-related processes. We use p53-deficient heterozygous male mice to examine the role of p53 in germ line mutations at ESTR loci. Males were irradiated with a variety of dose combinations both prior to and post-meiosis, and were mated to unirradiated wildtype females. DNA from the adults and offspring was analyzed for mutations at ESTR loci using DNA fingerprinting. Surprisingly, the study found no significant differences in germ line mutation rate between any treatment groups, including the 0Gy and 1Gy treatments. I discuss the possibility that these results are due to the p53 deficiency of the males, and that p53 homozygosity is necessary for radiation-induced germ line mutations at ESTR loci to occur. I conclude that further studies need to be done, including a control study using wildtype males of the same background strain as that of the p53 deficient line in order to verify our results.

abstract: Thesis

abstract: Master of Science (MS)


title: A Mixed Methods Study of Social Capital and Health Among Adults in Rural Ontario

contributor:
Valaitis, Ruta
Nursing

subject:
Social Capital
Rural Health

abstract: Social capital has shown the potential to benefit health, and therefore is an important concept to take up within nursing. However, the lack of consensus about how social capital should be defined and measured leads to challenges translating existing evidence into health promotion practice. Further, there is some literature suggesting that social capital may not benefit the health of rural residents in the same way as it does for urban residents. Therefore, there is a need for research that helps advance our conceptual knowledge of social capital while examining the concept and its impact on health for rural residents. This thesis involved a sequential explanatory mixed methods study to understand how rural residents experience social capital and how it impacts their health. In the first phase, I began with an exploratory factor analysis of the 2013 General Social Survey data. This revealed the underlying factors that made up social capital for urban and rural residents of Ontario. Logistic regression analysis indicated that four of the six social capital factors were positively associated with health. There were no differences between rural and urban residents in the factors revealed, nor in the influence of the factors on health, however rural residents scored higher on several social capital factors. In the second phase, interviews and focus groups in two rural Ontario communities helped explain the findings and explored how rural residents experienced social capital in their daily lives. The friendly and helpful social context helped elucidate why rural residents had high social capital scores, yet the structural context contributed to difficulties accessing social capital for some groups. Together, the data from both study phases help advance our knowledge of social capital with important implications for nursing practice.

abstract: Thesis

abstract: Doctor of Philosophy (PhD)

abstract: There has been a lot of attention to the topic of social capital and how it may benefit health. Social capital means the resources someone has access to because of belonging to a social network. This thesis aimed to understand what makes up social capital, how it influences health, whether there are differences in its impact between urban and rural residents, and how people living in two rural communities experience it in their daily lives. Quantitative analysis showed that some components of social capital benefited physical and mental health while others did not. Rural residents were not impacted any differently by social capital, however they had higher scores on several of its components than urban residents. Interviews and focus groups helped explain how the friendly and helpful social context of the rural environment contributed to high social capital, yet structural challenges meant some community members had difficulty accessing its benefits.


title: Studies on the Herpes Simplex Virus Type 1 gB Glycoprotein

contributor:
Ghosh, H. P.
Biochemistry

subject:
glycoprotein
herpes simplex virus
herpes
gB

abstract: The glycoprotein gB of HSV-1 is involved in viral entry and membrane fusion functions. It is glycosylated, forms homodimers, and is transported to both the inner nuclear membrane and plasma membrane in infected cells. The gB glycoprotein contains a potential membrane anchoring hydrophobic sequence of 69 amino acids, located near the carboxy terminus of the glycoprotein. This sequence is predicted to span the membrane bilayer three times, and can thus be divided into three distinct segments, each of which could span the bilayer. To define both the membrane anchoring sequence and the role of this 69 residue hydrophobic domain, a series of deletion mutants were constructed. These mutants have one, two or all three of the predicted membrane spanning segments deleted, in every combination possible, thereby creating a total of 7 deletion mutants. These mutant constructs were expressed in COS-1 cells using transient expression systems. All the mutant constructs were expressed and glycosylated in a manner similar to the wild type gB glycoprotein. Mutant glycoproteins lacking the third (or most carboxy terminal) predicted spanning segment of this 69 residue hydrophobic domain were found to be secreted from the cells, indicating that this segment may specify the membrane anchoring domain of the gB glycoprotein. Further, the mutant glycoproteins containing this third segment were localized in the nuclear envelope, while mutants lacking this segment were not. All the deletion mutants, except for one, were however defective in intracellular transport and processing of the N-linked oligosaccharides. The only mutant that showed any intracellular transport and processing had only the third segment deleted, but even this mutant was transported and processed much slower than the wild type glycoprotein. The mutant glycoproteins also failed to complement a gB-null virus. These results suggest that the carboxy terminus hydrophobic domain contains essential structural determinants of the gB glycoprotein.

abstract: Thesis

abstract: Master of Science (MS)


title: Transcription from HPV-16 Early Promoter (P₉₇) in an HPV-16/HSV-1 Recombinant Virus

contributor:
Smiley, James
Biology

subject: hpv virus transcription recombinant

abstract: Infection with Human papillomaviruses has been suggested to play an important role in the etiology of a number of human malignancies. The most investigated of HPVs is HPV-16. Infection with HPV-16, in association with other unknown factors, is implicated in the development of cervical cancer. Genetic analysis of HPVs has been hampered by the lack of an in vitro system in which the complete replicative cycle and the transcription of the various genes is possible. Replication and transcription of HPVs seem to be in part regulated by cellular factors expressed at different stages in the maturation of epithelial cells which constitute the normal hosts of those viruses. This study was primarily designed to address this difficulty. HPV-16 genome was introduced into a viral system, HSV-1, hoping that virally encoded factors can substitute for cellular factors required for the expression of HPV genes. This hypothesis was tested by analyzing the activity of the HPV-16 early promoter P₉₇ , in the constructed recombinant, in cells which are unsusceptible to infection with HPV-16. In Vero cells infected with the recombinant, P₉₇ was shown to be active. This suggests that HSV-1 encoded factors can influence transcription from endogenous papilloma promoters.

abstract: Thesis

abstract: Master of Science (MS)


title: Effects of the Den V Gene from the Bacteriophage T4 and the Human ERCC1 Gene on the Repair and Replication of Adenovirus in Mammalian Cells

title: Repair and Replication of Adenovirus in Chinese Hamster Ovary Cell DNA Repair Mutants

contributor:
Rainbow, Andrew
Biology

subject: bacteriophage repair mammal cell human gene ERCC1

abstract: The characterization of rodent cell mutants hypersensitive to UV light has led to the identification of at least 10 complementation groups all defective in some aspect of the first step in the excision repair of UV damaged DNA. The phenotypic properties of these mutants are thus of considerable importance to our understanding of DNA repair. In recent years five different excision repair cross complementing (ERCC) human genes have been isolated which correct the DNA repair deficiency in a number of Chinese Hamster Ovary (CHO) cell mutants and at least three of these genes also complement the repair deficiency in cells from patients suffering from xeroderma pigmentosum (XP), Cockayne syndrome (CS) and/or Trichothiodystrophy (TTD). Adenovirus (Ad) infection of rodent cells is generally semi-permissive and does not give rise to viral progeny, such that Ad reactivation in CHO cells has not previously been reported. This study utilizes the ability of CHO cells and human cells to replicate viral DNA in order to examine the reactivation of Ad in several CHO as well as human cell DNA repair mutants. Unirradiated and UV-irradiated suspensions of Ad were assayed for their ability to synthesize viral DNA following the infection of several CHO and human cell DNA repair mutants. The cell types examined included CHO cell mutants from complementation groups 1, 2, 3, 4, 5, 6, 9, 10 as well as human XP and tumour cells. The survival of viral DNA synthesis for UV-irradiated Ad was significantly reduced in several of the CHO and human cell mutants compared to that in normal cells. Cell mutants showing a reduced UV survival for this viral function included CHO cell mutants from complementation groups 1 to 6, XP cells and the 2 human tumour cell lines examined. This reduced host cell reactivation (HCR) for Ad indicates a reduced capacity for the repair of viral DNA in these cell types. DNA replication for unirradiated virus was also reduced for some of the mutants, especially the UV20 CHO cell mutant from complementation group 1, suggesting a deficiency for both DNA replication and repair in these cells. This study also used the recombinant viruses Ad5(denV) and Ad5(ERCC1) as vectors to examine the effect of the bacteriophage T 4 denV gene and the human ERCC1 gene on viral reactivation in the various cell mutants. UV survival of Ad5(denV) was increased compared to that of the control Ad5(LacZ) following infection of all the CHO and human cell types examined, indicating the denV gene product increases repair of Ad in both repair-proficient and repair-deficient cells. UV survival of Ad5(ERCC1) was increased compared to that of control Ad5(LacZ) following infection of the CHO mutant UV20 from complementation group 1, as well as all CHO cell types having normal HCR for AdS. However, UV survival of Ad5(ERCC1) was not increased compared to Ad5(LacZ) following infection of CHO mutants from complementation groups 2 to 6 and 10. These results support a specific complementation of the UV20 repair defect by ERCC1 and suggest that the human ERCC1 gene is more efficient than its hamster counterpart in repair-proficient CHO cells or that the ERCC 1 product is rate-limiting for the excision repair process in CHO cells. UV survival of Ad5(ERCC1) was also increased compared to Ad5(LacZ) in the normal human fibroblast cells and human tumour cells, but not in the XP (group D) cells. The kinetics of viral DNA synthesis and viral protein synthesis for unirradiated Ad5(denV) and Ad5(ERCC1) was also investigated following the infection of human and rodent cells. The deficiency in viral DNA synthesis and viral protein synthesis found for AdS(LacZ) following infection of rodent compared to human cells is partially complemented by either denV or ERCC1. The more marked deficiency in viral DNA synthesis of the UV20 CHO mutant was also complemented by either ERCC1 or denV, suggesting an ability of these genes to function in both repair and replication of viral DNA.

abstract: Thesis

abstract: Master of Science (MS)


title: Genotoxicity of 2-Nitrofluoranthene in Human Lymphoblastoid Cells In Vitro

contributor:
Tomkins, Darrell
Biology

subject:
gene
nitrofluoranthene
human
lymphoblastoid
in vitro

abstract: A micronucleus assay was developed using HSC-3TO, a human continuous lymphoblastoid cell line (LCL) and cytochalasin B (Cyt-B) to block cytokinesis. Mitomycin C (MMC), a chemotherapeutic agent and an inducer of micronuclei in various human cell types, was used as a positive control. MMC induced micronuclei in HSC-3TO at levels over six times that of control cells when cells were treated with the D₅₀ dose (i.e., the dose of MMC which was toxic to 50% of treated cells). It was found that harvest time was a very important variable and that a harvest time of 108 hours was optimal. 2-Nitrofluoranthene (2-NFA), a nitrated polycyclic aromatic hydrocarbon, the most abundant nitroarene found in the particulate organic matter fraction of ambient air and a potent direct-acting mutagen in the Ames Salmonella assay, was tested using the established CBMN assay protocol. Doses (20, 200 and 2000 ng/mL 2-NFA) were chosen based on the estimated yearly total body exposure and did not produce elevated levels of micronuclei at a dose of 100 times the estimated yearly total body exposure.

abstract: Thesis

abstract: Master of Science (MS)


title: The Phenotypic Impact of Mitochondrial DNA Mutations in Cancer

title: Mitochondrial DNA Mutations in Cancer

contributor:
Singh, G.
Biochemistry

subject: phenotype DNA mitochondria cancer

abstract: Mitochondrial DNA (mtDNA) aberrations have been detected in a large proportion of tissue samples isolated from human tumours, although the functional significance of these mutations is yet to be determined. Proponents of the selective advantage mtDNA defect acquisition model believe these mutations arise spontaneously, but propagate because they afford some benefit to overall neoplastic cell proliferation. The most tantalizing of these growth advantage properties is an elevation of electron transport chain (ETC) generated reactive oxygen species (ROS), which are already associated with some pathogenic mtDNA defects. However, others theorize that these mutations may arise spontaneously and expand through the ambivalent process of genetic drift alone. The 2008 human ovarian cancer cell line and its cis-platinum(II)-diammine-dichloride (CDDP) resistant C13* cell variant have divergent phenotypes when mitochondrial morphology, membrane potential (ΔΨₘ), ROS production, and rate of oxygen consumption are considered. Furthermore, the increased ΔΨₘ observed in C13* cells has been implicated in its CDDP resistant characteristic. In order to determine if mtDNA mutations were responsible for these phenotypic variations, the complete mitochondrial genomes of both cell lines were directly sequenced. Two novel mtDNA mutations were identified within cytochrome c oxidase subunit II (COXII) and the d-loop of 2008 and C13* cells respectively, however the functional significance of these defects were not obvious. To expunge the effects of nuclear DNA (nDNA) cybrids were created by transferring the mtDNA of 2008 and C13* cells to a common 143B TK⁻ nDNA background, creating 2008cyb and C13cyb cells. C13*cyb cells did not demonstrate CDDP resistance, decrease in rate of oxygen consumption or increase in ΔΨₘ when compared to 2008cyb cells. However, C13*cyb cells did retain mitochondrial morphological properties as well as increased Rh123 sensitivity and ROS production. This data would imply that mtDNA mutations are responsible for a proportion of mitochondrial-associated changes in the context of carcinogenesis.

abstract: Thesis

abstract: Master of Science (MS)


title: The Individual Roles of the Major E1B Proteins in Transformation and Their Function in the Lytic Cycle of Adenovirus Type 5

subject:
adenovirus
E1B protein
protein
transform
lytic cycle

abstract: Transformation by human adenovirus type 5 requires the cooperation of gene products from both the E1a and E1B early transcription units. Our major goal was to better understand the individual roles that the E1B proteins play in the transformation process. In order to determine the specific contribution made by the two major E1B proteins, 19K and 58K, mutants were constructed which were defective in the synthesis of each protein. Analysis with these mutants suggested that 58K appeared to be necessary for efficient plaque formation on human HeLa cells whereas 19K was not required. Mutants which failed to produce 19K or made a truncated 19K product displayed the cyt/deg phenotype characterized by production of large plaques and degradation of DNA These properties were not apparent with point mutants at methionine 120 or serine 164 of 19K or with mutants defective for 58K production. All E1B mutants produce E1A at levels comparable to wild type adenovirus 5, suggesting that neither E1B protein affects the regulation of E1A expression. Of interest was the observation that in combination with E1A, both 19K and 58K were able to induce transformation of baby rat kidney cells. However, the efficiency of transformation was greatly increased if both these E1B products were present. It seems likely that the mechanism of transformation involving each of these E1B proteins utilizes different pathways, but these pathways appear to be additive.

abstract: Thesis

abstract: Master of Science (MS)


title: Impact of Instruction on Preparation for Future Learning of Procedural Skills

contributor:
Grierson, Lawrence
Hunter, Andrea
Bridges, Ryan
Health Science Education

abstract:
Background: Competence in clinical practice involves effective application of previously-acquired knowledge and the ability to use knowledge adaptively for innovation across diverse clinical contexts. In the learning context, this adaptability is termed Preparation for Future Learning (PFL) and looks at the capacity of trainees to use knowledge and strategies to learn and solve new problems. We compared how three instructional designs impacted procedural skill acquisition and PFL, through assessment of transfer to learning a novel procedural skill.
Methods: We randomized 60 medical students to practice infant lumbar puncture (ILP) according to a Simulation-based Discovery Learning intervention (i.e. guided invention before expert instruction), a Simulation-based Mastery Learning condition (i.e., a stringent form of competency-based education), or a control sequence. In a second session, we assessed how well learners transferred strategies developed in the first session to learn a novel task – knee arthrocentesis (KA). We compared trainees’ post-test ILP performance and PFL ability via a KA performance test.
Results: There was a significant effect of group on ILP checklist score after controlling for pre-test score, F(2,56) = 3.202, p<0.05. Post-intervention ILP checklist scores were statistically greater in the mastery group (93.03% ±2.02) compared to the control group (85.94% ±1.93). There was no significant effect of group on ILP global rating score. Though reliability of ratings was low, there was no significant effect of group on KA performance.
Conclusion: This work supports a benefit of mastery learning compared with non-mastery simulation-based instruction for procedural skill acquisition, but did not demonstrate a benefit for mastery learning over invention followed by direct instruction. Most participants in all groups were not competent to perform KA and no intervention showed clear benefit for PFL. Future work is required to guide development of learning interventions which support PFL given the expectation for trainees to problem solve and innovate.

abstract: Thesis

abstract: Master of Science (MSc)


title: Abrasion Assisted Wire Electrical Discharge Machining

contributor:
Koshy, Philip
Mechanical Engineering

subject: abrasion electric machining

abstract: The adoption of Electrical Discharge Machining (EDM) technologies to mainstream manufacturing saw dramatic advances in the process starting in the 1980's. Wire Electrical Discharge Machining (WEDM) in particular achieved enhancements in cutting speed of over 800% between 1980 and 1992 due to improvements in generator and wire technology. Since then, increases in cutting speed have been gradual. To achieve dramatic improvements to the process once again, a paradigm shift, from improving upon existing technologies to introducing and developing new one is required. In this light, an investigation into the proof-to-concept and development of a novel hybrid machining process based upon Wire Electrical Discharge Machining (WEDM) and abrasive technologies is presented. In the process termed Abrasive Wire Electrical Discharge Machining (AWEDM), material removal is shown to occur by the simultaneous action of electrical erosion and abrasion. Through experimental evaluation, this combination is shown to bring about a manyfold improvement in the material removal rate and to generate machined surfaces with minimal recast layer, in comparison to conventional WEDM processes. To understand the operation of the process and to control the proportion of abrasion and EDM taking place, the effect of varying the process conditions is studied. The servo-reference voltage and peak discharge current, in particular, provide effective means to control the process. Practical implementation of the process presents several challenges, such as accurately guiding an abrasive wire; a discussion of some of these issues and possible solutions is included. The need for a wire that is specially suited to AWEDM is demonstrated with a discussion of the requirements and possible designs for such a wire. Whether or not a manufacturing process sees practical industrial use is chiefly dictated by economics. By considering the increase in both productivity and wire cost, AWEDM is shown to be economically feasible and offer potentially substantial benefits. This work ultimately serves as the basis for future work with respect to AWEDM. The work herein covers a broad range of topics in hopes of guiding future areas of research.

abstract: Thesis

abstract: Master of Applied Science (MASc)


title: Regulation of HSV-1 Immediate Early Gene Expression

subject:
herpes
herpes simplex virus
gene
expression
regulate

abstract: Herpes simplex Virus Type 1 expresses three different classes of genes, immediate early, early, and late, during a lytic infection. Immediate early genes are the first class of genes expressed and they are the only genes expressed independently of de novo viral protein synthesis. This unique characteristic is thought to be the result of the activation of immediate early genes by Vmw65, a protein brought into the cell as a component of the infecting virion. Vmw65 transactivates through the target sequence TAATGARAT (R= purine) which is present at least once in all immediate early transcription regulatory regions. By inserting minimal synthetic promoters, containing the TAATGARAT sequence, into the thymidine kinase locus of the herpes simplex virus type 1 genome I determined that transactivation by Vmw65 is not sufficient to confer on the linked sequences the complete immediate early pattern of gene expression. Furthermore through a transient expression assay I determined that the TAATGARAT sequence element, by itself, when linked to a TATA box is sufficient to act as a target for Vmw65 transactivation.

abstract: Thesis

abstract: Master of Science (MS)


title: Modulation of Peroxisome Proliferator-Activated Receptor α Activity by Mitochondrial 3-Hydroxy-3-Methylglutaryl Coenzyme A Synthase

title: Modulations of PPARα Activity by mtHMG-CoAS

contributor:
Capone, John
Biochemistry

subject: peroxisome receptor mitochondria coenzyme

abstract:
The regulation of gene expression at the level of transcription is an important mechanism for maintaining homeostasis. The peroxisome proliferator-activated receptor α (PPARα) is a member of the nuclear hormone receptor superfamily that is involved in transcriptionally modulating such pathways as lipid and fatty acid metabolism. This receptor binds to enhancer elements, peroxisome proliferator-responsive elements (PPREs), upstream of a variety of target genes including those involved in β-oxidation of fatty acids, in the peroxisome and mitochondria, and ketogenesis. One such element has been identified upstream of the rat mitochondrial 3-hydroxy-3-methylglutaryl coenzyme A synthase (mtHMG-CoAS) gene. This enzyme has been shown to be one of the key regulatory points of ketogenesis.
To learn how PPARα mediated transcriptional regulation occurs, this receptor was used as bait in a yeast dihybrid screen and was found to interact with human mtHMG-CoAS. Reproduction of this interaction 𝘪𝘯 𝘷𝘪𝘵𝘳𝘰 was performed by solid phase capture assays, using GST fusion proteins, and by co-immunoprecipitations. It was also ascertained that the synthase enzyme interacts with the retinoid X receptor α (RXRα). The hamster cytoplasmic form of the enzyme was chosen as a control and showed no binding capabilities to either nuclear hormone receptor. Interestingly, the mitochondrial enzyme contains a motif, LXXLL that has previously been shown to be important for binding between a transcriptional coactivator and a receptor. A site-directed mutant of the mitochondrial synthase sequence from
LASLL to LASVL was made. The mutant showed a reduced ability to interact with both nuclear receptors. Consequently, the LXXLL motif is responsible, at least in part, for the interaction between mtHMG-CoAS and both PPARα and RXRα. The cytoplasmic synthase does not contain the motif; its corresponding sequence is LASVL. The effect ofthe mitochondrial synthase within a cell was then determined by transient transfection assays. It was discovered that on the HMG PPRE the mitochondrial synthase potentiated PPARα mediated transactivation while on the AOx PPRE the enzyme inhibited it. Thus, mtHMG-CoAS modulates PPARα activity in a PPRE dependent manner. The LXXVL mitochondrial mutant inhibited PPARα transactivation on both the HMG and AOx PPRE. Therefore, the mutant acts as a
dominant-negative inhibitor of α mediated activity. The cytoplasmic control enzyme had no effect on either PPRE. To determine if the mitochondrial enzyme could be detected within the nucleus where it appeared to be modulating PPARα mediated transcription, localization studies were performed with the use of immunofluorescence. Immunofluorescence was done by utilizing hemagglutinin (HA) epitope tagged fusions of the mitochondrial, mutant and cytoplasmic enzymes. When the HA tag was placed at the carboxyl terminus of mtHMG-CoAS, the enzyme was localized to the mitochondria with no apparent nuclear staining. This is consistent with previous localization studies done with the rat mtHMG-CoAS. Also, the mutant enzyme, with the HA tag at the carboxyl terminus, was only detected in the mitochondria. However, under certain conditions, when PPARα and the mitochondrial synthase were co-transfected, the mitochondrial enzyme was detected within both the mitochondria and the nucleus. The mutant, on the other hand, when co-transfected with PPARα was found to remain non-nuclear. The HA tagged cytoplasmic control enzyme was also non-nuclear. Therefore, mtHMG-CoAS can be detected within the nucleus, it binds, due to at least in part a LXXLL motif, to nuclear receptors, and it is capable of modulating transcription in a PPRE dependent manner. Ketogenesis becomes an important mechanism for fuel production during starvation, prolonged exercise and diabetes. Perhaps, during extreme circumstances such as starvation, mtHMG-CoAS is involved in autoregulation which allows for an amplification of the transcription of its own gene. Also, mtHMG-CoAS appears to inhibit the transcription of the AOx enzyme which is involved in peroxisomal fatty acid β-oxidation. Again, perhaps under extreme conditions, the β-oxidation of fatty acids is concentrated within the mitochondria which allows for the production of acetyl-CoA that can subsequently be converted to ketone bodies that can then be utilized for fuel.

abstract: Thesis

abstract: Master of Science (MS)


title: Studies on the Role of the Herpes Simplex Virus ICP4 Protein in Adenovirus Gene Expression

title: An Adenovirus Type 5 Recombinant Vector Encoding the HSV-1 Protein, ICP4

contributor:
Bacchetti, S.
Biology

subject:
herpes
herpes simplex virus
ICP4
protein
adenovirus
gene

abstract: Many viral transcriptional activators have been shown to activate genes of heterologous systems. To assess the ability of the herpes simplex virus ICP4 trans-activating protein to complement an adenovirus mutant lacking its own trans-activator, the E1a protein, I constructed an adenovirus type 5 vector containing a temperature sensitive ICP4 gene, under control of its own promoter, within the E1 region of the genome. The recombinant virus expresses ICP4 in human cells which are permissive (293) or nonpermissive (KB and R970-5) for E1a⁻ viral replication, and at levels which approximate those obtained in herpes simplex infection. The adenovirus encoded protein is functional in that it complements an ICP4 deletion mutant of herpes simplex virus, however it is incapable of complementing adenovirus E1a⁻ mutants for viral growth or DNA replication. At the level of activation of gene expression, ICP4 stimulates the expression of the adenovirus E2a gene but not that of other early genes. My results indicate that ICP4 does not possess all of the functions of the E1a proteins and, furthermore, that adenovirus early genes differ in their susceptibility to heterologous trans-activators.

abstract: Thesis

abstract: Master of Science (MS)


title: Identification of the SV40 Gene Product(s) Required for Induction of Chromosomal Aberrations in Human Cells

contributor:
Bacchetti, Silvia
Biology

subject: gene SV40 chromosome cell aberration induction

abstract: Expression of the Simian Virus 40 (SV40) early region in human cells results in the induction of chromosomal aberrations and polyploidy, and in transformation. To understand how genetic damage occurs and what role it plays in transformation, human diploid fibroblasts and embryonic kidney cells were transfected with plasmids encoding wild type or mutant forms of the viral early region, and the neo gene. Clones selected for G418 resistance and expressing viral genes were initially analyzed within 20 cell divisions. The results of this study demonstrate that expression of the SV40 large T antigen is sufficient for the induction of chromosomal damage and ploidy changes, and that small t does not contribute to these processes. Mutant plasmids either lacking the SV40 origin of DNA replication, or encoding a large T mutant defective in its ability to bind the retinoblastoma gene product (Rb) were as proficient as wild type plasmids, indicating that both viral DNA replication and binding of T antigen to Rb are not required for cytogenic damage. On the other hand, preliminary results with a plasmid encoding a T antigen mutant unable to bind the cellular p53 protein suggest that formation fo this complex may be important for cytogenetic damage. This study has also shown that chromosomal aberrations, but not necessarily polyploidy, increase in frequency and complexity upon subculturing of the clones regardless of whether such populations arrest at crisis or yield immortal lines. These results are compatible with the hypothesis that large T antigen destabilizes the cellular genome, and that specific mutations arising from this process may contribute to cell immortalization.

abstract: Thesis

abstract: Master of Science (MS)


title: Subcellular Localization of the HSV-1 Proteins VHS and VP16

subject:
herpes simplex virus
herpes
protein
subcellular

abstract: Infection of a host cell by the Herpes Simplex Virus Type 1 leads to the efficient reprogramming of the cells' synthetic machinery to replicate the viral genome ultimately producing progeny virions. Two proteins introduced upon viral fusion are thought to initiate this effect. The potent transactivator of immediate early genes (VP16) and the mRNA destabilizing virion host shutoff protein (vhs), work in concert with one another to invoke the cascade of viral gene expression, and to destroy pre-existing cellular mRNA. Due to the non-specific nature of vhs induced mRNA degradation, its activity is downregulated at later times during infection to spare virally encoded mRNA. Recent evidence has shown that VP16 is responsible for this vhs downregulation, a process thought to occur by mutual interactions between the two proteins and a potential compartmentalization of vhs within the nucleus (Lam 𝘦𝘵 𝘢𝘭., 1996; Smibert 𝘦𝘵 𝘢𝘭., 1994). Furthermore, such an event is also thought to position vhs so it can be efficiently packaged, a supposition supported by the observation that vhs lacking the ability to bind VP16 is not incorporated into new virions (Read 𝘦𝘵 𝘢𝘭. , 1993). To ascertain if VP16 was indeed capable of relocalizing vhs to the nucleus of a cell in the absence of any other viral factors, we created multiple constructs consisting of various portions of vhs fused in frame to the fluorescent marker protein EGFP. In addition, various truncated forms of VP16 were also fused to EGFP for the purpose of delineating the region of VP16 that is responsible for VP16 and possibly vhs nuclear localization. Co-transfection experiments utilizing EGFP-vhs fusions demonstrated that vhs relocalizes to perinuclear regions in the presence of VP16, an effect absolutely dependent upon its ability to interact with VP16. In addition, deletion mapping of VP16 implicated the region spanning amino acids 335 to 355 as being necessary for this localization, with a stretch of 15 amino acids (330 to 344) appearing to constitute a putative bipartite nuclear localization signal. Interestingly, our observation that the vhs/VP16 complex localizes to a region of the cell thought to ultimately encompass the tegument of new virions gives credence to the notion that this interaction and subsequent localization may indeed function to package vhs into new virions. Furthermore, it is also suggested that vhs may in fact be downregulated at intermediate times during infection through VP16 mediated compartmentalization within the nucleus. For these reasons we propose that the disruption of the vhs/VP16 interaction could severely abrogate the infectivity of HSV and as such could present a novel target for antiviral intervention.

abstract: Thesis

abstract: Master of Science (MS)


title: Diet of Pumpkinseed Sunfish (Lepomis gibbosus) and Brown Bullhead (Amerius nebulosus) in the Littoral Zone of Hamilton Harbour

title: Fish Feeding in Hamilton Harbour

contributor:
Kolasa, Jurek
Biology

subject: sunfish diet harbour bullhead

abstract: Benthic macroinvertebrates, pumpkinseed sunfish (𝘓𝘦𝘱𝘰𝘮𝘪𝘴 𝘨𝘪𝘣𝘣𝘰𝘴𝘶𝘴) and brown bullhead (𝘈𝘮𝘦𝘳𝘪𝘶𝘴 𝘯𝘦𝘣𝘶𝘭𝘰𝘴𝘶𝘴) were sampled at three sites in western Hamilton Harbour and Cootes Paradise in early June 1995. The diversity, abundance, similarity and variability of the substrate benthic community and the gut contents of the fish were analyzed. The gut contents of both species of fish reflected the diversity, similarity and variability of the site at which they were caught. Mean gut fullness, analysized using prey abundance and volume, shows no significant difference between sites, suggesting that the fish were eating similar amounts of prey at all three sites. Both pumpkinseed sunfish and brown bullhead fed selectively on certain size classes and benthic taxa, including several taxonomic groups previously unreported for these species. Differences in benthic community structure at the three sites and the resultant differences in fish diet have important implications for the remediation of Hamilton Harbour. As water quality improves and the macrophyte cover increases, benthic diversity and abundance will increase. These improvements will increase the area of the littoral zone and the quality of the rearing and feeding environments for the recovering warmwater fisheries in Hamilton Harbour.

abstract: Thesis

abstract: Master of Science (MS)


title: Mechanisms of Action of and Cellular Resistance to Chemotherapeutic Agents in Human Cells: Possible Applications to Quantitative Mutagenesis

contributor:
Gupta, R. S.
Biochemistry

subject: cell resistance chemotherapy human mutagenesis

abstract: The aim of this study was to investigate the mechanisms of action of and development of cellular resistance to various anticancer agents in human (HeLa) cells using a combined genetic and biochemical approach. The agents employed for this purpose included: the purine nucleoside analogues, toyocamycin, tubercidin, and 6-methyl-mercaptopurine riboside (6-MeMPR); the protein synthesis inhibitor, puromycin; and the microtubule stabilizer, taxol. To investigate the mechanisms of action and cellular resistance to the purine nucleoside analogues, stable first-step toyocamycin, tubercidin and 6-MeMPR resistant HeLa mutants were isolated. These mutants exhibited high degrees of resistance and cross-resistance to various adenosine kinase-activated nucleoside analogues, possessed <2% of the adenosine kinase activity of parental HeLa cell extracts and exhibited severely reduced cellular uptake and macromolecular incorporation of adenosine in vivo. These results indicate that in human cells the cytotoxic effects of toyocamycin, tubercidin and 6-MeMPR are dependent upon adenosine kinase-catalyzed phosphorylation of these drugs to their respective monophosphates and that resistance to these agents results from a deficiency in adenosine kinase activity in vivo. Further insight into the nature of the genetic and biochemical alteration(s) affecting adenosine kinase in these mutants was achieved using SDS-polyacryamide gel electrophoretic and immunoblot analysis. Immunoblots -revealed that each toyocamycin, tubercidin and 6-MeMPR resistant mutant contained similar amounts of cross-reacting material that had the same electrophoretic mobility as adenosine kinase in parental HeLa cells. Therefore, the lesion in these mutants must be a missense type of alteration in the structural gene for adenosine kinase. The utility of the 6-MeMPR resistant mutant selection system for quantitative mutagenesis studies was also investigated. Numerous favourable attributes appropriate to mutagenesis studies were found using this selection system. These included: the obtainment of highly resistant mutants which were stable in the absence of drug, the absence of cell density or cross-feeding effects in the selection system, maximum phenotypic expression required a relatively short time period and mutagen treatment increased the mutant frequency in a linear dose-dependent manner. Thus, selection for genetic alterations at the adenosine kinase locus appears to provide a valuable system for quantitative mutagenesis studies in human cells. The combined genetic and biochemical approach was also used to investigate the development of resistance to puromycin and taxol. Therefore, first-and second-step mutants resistant to each of these drugs were selected and characterized. Cross-resistance and uptake studies with the puromycin resistant mutants suggest that the most common -mechanism for the development of cellular resistance to puromycin in human (HeLa) cells involves an alteration in membrane permeability that reduces drug uptake/transport. Similar studies with the taxol resistant mutants suggest the existence of two possible mechanisms for the development of resistant to this agent in human (HeLa) cells. One mechanism involves a biochemical lesion that specifically affects a microtubule-related cellular component. The second mechanism, however, nonspecifically affects cellular membrane permeability and results in reduced drug uptake/transport.

abstract: Thesis

abstract: Master of Science (MS)


title: Partnering with families to mobilize a holistic family-centred approach to childhood disability: A multi-faceted integrated knowledge translation project

contributor:
Rosenbaum, Peter
Rehabilitation Science

subject: knowledge translation

subject: integrated knowledge translation

subject: childhood disability

subject: International Classification of Functioning, Disability and Health

subject: family-centred service

subject: family engagement in research

subject: child health

subject: knowledge to action framework

abstract:
Background: This thesis aimed to develop, implement, and evaluate a multi-faceted integrated knowledge translation (iKT) intervention to disseminate and support adoption of the ‘F-words in Childhood Disability’. Grounded in the WHO’s ICF framework, the ‘F-words’ (Function, Family, Fitness, Fun, Friends, and Future) offer a holistic family-centred approach to childhood disability.
Methods: This thesis was guided by the action cycle of Graham et al.’s (2006) knowledge-to-action (KTA) framework. Chapter 2 reports a knowledge translation (KT) initiative (i.e., an online video) to disseminate the ‘F-words’ and explore people’s reception of these ideas. Chapter 3’s scoping review identifies and assesses KT strategies that directly target families raising children and youth with special health care needs. Chapter 4 describes a pilot study to evaluate the usability and utility of an online ‘F-words’ KT resource. Chapter 5 reports a case study of our longitudinal KT research program, and uses Diffusion of Innovation (DOI) theory to understand the factors that contributed to the adoption of the
‘F-words’.
Results: Multi-faceted KT strategies, co-developed with stakeholders, were essential to moving the ‘F-words’ into practice. The video (Chapter 2) reached >700 views in two months and 98% of 137 survey respondents indicated they ‘extremely liked’/‘liked’ the ideas. The scoping review (Chapter 3) identified six studies, all of which evaluated educational materials and deemed them to be useful and important to families. The pilot evaluation (Chapter 4) revealed the online resource to have positive usability and utility for families and service providers. The case study (Chapter 5) highlighted that diffusion, dissemination and implementation strategies were all needed and that DOI factors (i.e., the innovation characteristics, communication channels, social networks, and time) contributed to the adoption of the ‘F-words’. Based on a multi-faceted integrated KT research program we now have extensive examples of ‘F-words’ adoption by families, service providers, and health care organizations.
Conclusions: This thesis illustrates a step-wise theory-informed approach to the development and evaluation of a multi-faceted iKT intervention. By studying each step of the action cycle, this work contributes new knowledge to both the processes involved in disseminating research evidence, and associated outcomes from a multi-faceted iKT intervention. Findings from this thesis contribute new discoveries to both KT practice and science.

abstract: Thesis

abstract: Doctor of Philosophy (PhD)

abstract: In the 21st century, research supports a holistic family-centred approach to childhood disability. Unfortunately, a research to practice gap remains, and the professional-led biomedical approach still informs many practices. In 2012, Rosenbaum and Gorter published “The ‘F-words’ in childhood disability: I swear this is how we should think!” They highlighted the importance of ‘Function, Family, Fitness, Fun, Friends, and Future’, grounded in the World Health Organization’s framework for health. This thesis developed and evaluated a knowledge translation research program to move the ‘F-words’ into practice. Objectives, all achieved, were to: i) apply strategies to spread awareness of the ‘F-words’ and explore people’s reception of these ideas; ii) identify and assess strategies to share research with families; iii) develop and evaluate an online resource to support use of the ‘F-words’; and iv) study the processes involved and factors that contributed to the ‘F-words’ adoption. These findings have implications for both doing and studying knowledge translation.


title: Experimental and Numerical Studies of Deep Drawing and Redrawing of 304 Stainless Steel Sheet

contributor:
Jain, Mukesh
Mechanical Engineering

subject:
experimental
drawing
redrawing
stainless steel
study

abstract: The drawability of sheet material in deep drawing followed by redrawing has not been studied theoretically in detail. The clamping force during deep drawing has not been properly simulated by FE method in the past due to the neglect of the operating machine stiffness. In addition, deep drawing process with centre hole blank has not been investigated in detail experimentally and numerically in the previous work. All of these aspects are studied in this work. A new FE model for deep drawing and redrawing has been developed, which accounts for the operating machine stiffness. In this model, the draw die is connected to a semi-rigid component of the test frame by some spring elements so that the stiffness of the operating machine can be controlled by the stiffness of these springs. Also, a mathematical model to determine the limiting drawing ratio (LDR) of deep drawing and redrawing processes has been derived based on the extension of an existing analytical model and Hill's anisotropic criterion. The results of the mathematical model have been validated by corresponding experimental and FE simulation work in terms of punch load and clamping force versus punch displacement and thickness distributions along the product profile. Furthermore, deep drawing with centre hole blank has been studied in terms of flanging ratio and the results of FE simulation are in good agreement with experimental work.

abstract: Thesis

abstract: Master of Applied Science (MASc)