title: Atrial Natriuretic Peptide and its Possible Role in Post Exercise Hypotension

contributor:
MacDougall, J. D.
Human Biodynamics

subject: natriuretic post-exercise hypotension peptide

abstract: The mechanisms which cause post exercise hypotension (a phenomenon of prolonged, decreased resting blood pressure following physical exertion) are unknown. Atrial natriuretic peptide (ANP) is known to exert potent natriuretic and vasodilatory properties which play an integral role in fluid regulation and blood pressure control. Elevations in plasma ANP concentration have been shown to occur during dynamic endurance exercise, and to a lesser extent during heavy resistance exercise. The purposes of this investigation were to 1) examine the effects of resistance and endurance exercise on the release of ANP, 2) examine the effects of resistance and endurance exercise on post exercise blood pressure and 3) evaluate the potential correlations of ANP release with any observed changes. Thirteen males (24.3±2.4yrs.) performed 15 min of unilateral leg press (65% 1 RM) and, one week later ~15 min (based on summed cardiac cycles of the resistance trial) of cycle ergometry (65% V0₂ ₚₑₐₖ). Blood pressure was measured using an intra-arterial catheter during exercise and for 1 h post exercise. Arterial blood was drawn at rest, 5, 10 and 15 min of exercise and 1 1/2, 3, 5, 10, 15, 30, 45 and 60 min post exercise for subsequent analysis of hematocrit and αANP. No differences occurred in blood pressure responses between trials, but significant decrements in blood pressure occurred post exercise compared to pre exercise. Systolic pressure was ~20mmHg lower from 10 min post exercise until measurements terminated at 60 min post exercise. Mean pressure was also significantly attenuated by ~7 mmHg from 30 min post exercise onwards. Only slight (non significant) elevations in αANP concentration were detected immediately following exercise with no elevation present by 5 min post exercise. It was concluded that post exercise hypotension occurs with acute bouts of either resistance or endurance exercise and that αANP does not appear to be directly related to this hypotensive effect. This study was supported by the Natural Sciences and Engineering Research Council of Canada

abstract: Thesis

abstract: Master of Science (MSc)


title: The Characterization of a Novel Putative Signalling Protein and its Interaction with Tyrosine 1253 of the NEU/ERBB2 Receptor Tyrosine Kinase

title: The Characterization of the p34-NEU/ERBB2 Interaction

contributor:
Muller, William
Biology

subject:
protein
tyrosine 1253
NEU/ERBB2
tyrosine kinase

abstract: The Neu/ErbB2 receptor tyrosine kinase has been implicated in the induction of mammary tumourigenesis. Both Ras-dependent and independent signalling downstream of activated Neu is believed to mediate cellular signalling that contributes cellular transformation in oncogenic Neu. Signalling from five 𝘣𝘰𝘯𝘢 𝘧𝘪𝘥𝘦 autophosphorylation sites (termed sites A through E) in the carboxyl tail of the receptor mediate these signals to the cytosol. Of the four positive regulatory sites (B, C, D, E), only three (B, C, D,) signal through known signalling molecules. Site E, (Y1253) in Neu, though known to interact with DOKR is essentially an orphan site. The discovery of a 34kD protein capable of associating to peptides corresponding to site E prompted investigations into the nature of site E signalling. Mass spectrometric analyses revealed that the 34kD protein is 2,4-dienoyl-CoA reductase (DECR1), a lipid metabolism protein typically localized to the mitochondria. Investigations into this protein reveal that DECR1 is capable of associating with Neu site E in a tyrosine phosphorylation and sequence specific manner. Furthermore, analyses with site E second-site mutants (YE[APEY], YE[DPEY], YE[NAEY] and YE[NDEY]) show that DECR1 associates in a manner consistent to a PTB domain-containing protein. Analyses of amino acid sequence demonstrate the presence of a putative Bcl-domain in DECR1 suggestive of a role in apoptosis. Experiments into the apoptotic activity of DECR1 proved inconclusive. The examination of sub-cellular localization of Neu and DECR1 showed that the two proteins are found in both the mitochondrial and plasma membrane fractions. These results demonstrate that DECR1 may be a veritable binding partner for Neu Y1253.

abstract: Thesis

abstract: Master of Science (MSc)


title: Investigations of the Active Site of Microsomal Leucine Aminopeptidase by Probing with Ethylenediamine Derivatives

contributor:
Chan, William
Biochemistry

subject:
microsomal leucine
aminopeptidase
ethylenediamine

abstract: The active site of porcine microsomal aminopeptidase was probed by studying the inhibition of the enzyme using derivatives of ethylenediamine and diaminopropionic acid. In addition, some amino acids, substituted hydroxamates and phosphates were also tested. In order to synthesize diaminopropionic acid derivatives, CBZ-amino acid p-nitrophenyl esters were reduced to the corresponding aldehydes by lithium tri-t-butoxy-aluminohydride. Through the Strecker synthesis, the aldehyde intermediates were converted to diaminopropionitrile analogues which were then hydrolysed in acid to the desired products. Unfortunately, these compounds were not potent inhibitors for this enzyme. α-Amino acids were found to be better inhibitors than their β-amino counterparts and the Kᵢ of α-leucine was about 7-fold lower than its B-analogue. The amino group position of the amino acids is therefore important for enzyme recognition. On the other hand, N-alkylation of ethylenediamine was observed to abolish its inhibition potential. Furthermore, another unexpected finding in this work is that N- or 0-methylation of the hydroxamate group hinders the ability of these inhibitors to act as a bidentate zinc ligand. Although some phosphate derivatives that we tested showed poor inhibitory potency, phosphonamidate, a potential transition state analogue, might serve as a powerful inhibitor. In summary, the relationship between the structure and inhibitory potency of some inhibitors was demonstrated.

abstract: Thesis

abstract: Master of Science (MSc)


title: Gene Expression Analysis for Time-Course Microarray Data

contributor:
Esterby, Sylvia
Statistics

subject: gene time-course data

abstract:
DNA microarray technology makes it possible to analyze the expression levels of many thousands of genes simultaneously. One of the goals of microarray data analysis is to understand the multiple biological roles of genes and their interactions in complex biological processes. Genes with similar expression patterns are likely to share similar functions or biological processes. Therefore, analysis of changes in gene expression of a certain biological processes over time is of particular interest. Unsupervised clustering methods provide an efficient way of finding overall patterns and tendencies by clustering microarray gene expression data. The genes in the same cluster are regulated in a similar manner based on the assumption above. But traditional unsupervised clustering methods usually end up with clusters of genes with similar expression patterns but without interpretations describing the clusters in terms of gene functions or processes involved.
In this project, some statistical techniques are applied to analyze the data set from microarray experiments of sporulation in yeast. These techniques include LOWESS data normalization, which is intended to remove the systematic variations from the data; a partitional clustering method, K-means, is used with initial centroids obtained from hierarchical clustering method of DIANA; the "gap statistic" technique is implemented to estimate the "optimal" number of clusters in the data set; and finally multiple hypothesis testing is used to determine whether biologically related genes are statistically over-represented in the gene clusters using the web query tool FatiGO. These methods are combined with graphical representation of cluster profile shape and colour maps of up and down regulation via heat maps. Application of these methods to a yeast sporulation time-course data set [Chu 𝘦𝘵 𝘢𝘭. 1998] demonstrates the utility of cluster analysis to such data sets and provides an automated method for including biological information about gene function and characteristics.

abstract: Thesis

abstract: Master of Science (MSc)


title: An Interactive System for Sequence Analysis

contributor:
Jiang, Tao
Computation

subject: sequence analysis

abstract: Sequence Analysis Tool (SAT) is an X-window (OPEN LOOK version) based interactive system developed for sequence analysis. In this first version, it provides a friendly graphical user interface and convenient functions for performing various tasks required in sequence alignment. In particular, space-efficient algorithms for pairwise alignment and 3-star alignment are implemented as functionalities, which can be used to serve most sequence alignment tasks and therefore provide a basis for further improvement of this tool. SAT is also targeted at providing a testing platform for performance analysis of various alignment-related algorithms. A set of procedures is developed to provide an application programming interface with which other related programs can be easily connected to SAT. SAT is programmed in C/Xlib/OLIT. The object-oriented style makes further maintenance and improvement easy.

abstract: Thesis

abstract: Master of Science (MSc)


title: Effects of O^6-Alkyl Guanosine Residues on RNA Duplex Stability

title: O^6-Alkyl Guanosine Residues in RNA Duplexes

contributor:
Neilson, Thomas
Biochemistry

subject: alkyl guanosine RNA stability

abstract: Several short oligoribonucleotide sequences containing modified purine residues of biological significance were synthesized using the phosphotriester method developed in Neilson's laboratory. Variable temperature proton nuclear magnetic resonance (NMR) spectroscopy was used to examine the solution conformations of these oligomers. Studies of the effect of position, type and extent of alkyl modifications on helix structure and stability were undertaken. The triribonucleotide GpCpA was the first trimer shown to form a stable RNA duplex (Tm 33°C) (Alkema, et al, 1981(a)). This duplex contained two G:C base pairs and two 3'-dangling adenosine residues, and had a stability equal to that of the tetramer duplex UpGpCpA, having four Watson Crick base pairs. A series of GpCpN trimers was prepared (N= m⁶A, m⁶₂A, m¹G, m⁶G, e⁶G, m²m⁶G), using GpCpA as a reference, to determine how N-or O-alkylation of the dangling residue affected duplex stability. Both the studies of the N-alkylated (N=m⁶A, m⁶₂A, m¹G) (D'Andrea, et al, 1983) and 0-alkylated (N=m⁶G, e⁶G, m²m⁶G) sequences (present work) led to the conclusion that site and degree of modification were important factors for stability. Comparison of N-versus O-alkylated sequences revealed how the hydrophobic regions surrounding the alkylated nitrogen or alkylated oxygen atoms, and the spatial location of these regions, contributed to duplex stability. Examination of the effect of modified guanosine residues within a short squence, was performed through studies on ApGpNpCpU pentamers (N=m⁶G, e⁶G, m²m⁶G), having N in internal non-base-pairing and. in internal base-pairing positions. No duplex formation was seen, in contrast to studies involving reference compounds : ApGpGpCpU (a qualitative reference), ApGpGpCpU : ApGpUpCpU (Tm 31.4 C), and ApGpGpCpU : ApGpCpCpU (Tm 47.0 C). As no melting temperatures could be calculated for the modified strands, and because their NMR analyses were so similar, no comparisions regarding degree of destabilization, could be made amongst the various modified residues. Never the less, it is clear that O-alkylation of the central G residue significantly disrupts duplex formation, through generation of a centre of great instability. This result sharply contrasts that when the same modified residues are located in terminal, non-bonding positions.

abstract: Thesis

abstract: Master of Science (MSc)


title: Creation and Characterization of a Fluorescence Signalling DNA Enzyme

title: A Fluorescence Signalling DNA Enzyme

contributor:
Li, Yingfu
Biochemistry

subject:
fluorescence
DNA
enzyme
in vitro

abstract: 𝘐𝘯 𝘷𝘪𝘵𝘳𝘰 selection has been widely used to isolate single-stranded DNA molecules from large random-sequence pools that are able to perform a desired catalytic reaction, or bind to a target molecule. Using this technique, we have created a DNA enzyme, named DET22-18, which has a uniquely linked chemical catalysis/real-time signaling capability. It is a true enzyme with a 𝘬cₐₜ of~7 min⁻¹ -the second fastest rate ever reported for a DNA enzyme. The DNA enzyme cleaves a substrate containing a single ribonucleotide linkage embedded in a DNA chain and sandwiched between a fluorophore-labeled deoxyribonucleotide and a quencher-modified deoxyribonucleotide. The ability of DET22-18 to generate a large fluorescence signal provides a useful tool to engineer potential allosteric deoxyribozyme biosensors for real-time detection of important biological targets. To provide a proof of concept that a reporter system can be built from the above signaling DNA enzyme, the cis-acting version of this enzyme, DEC22-18A, was engineered into an allosterically regulated deoxyribozyme biosensor that can report ATP. A preliminary investigation was also conducted to determine a possible secondary structure of the DNA enzyme. This study lays a foundation for pursuing novel signaling DNA enzymes for biological detection directed applications.

abstract: Thesis

abstract: Master of Science (MSc)


title: Anaerobic Energy Supply During Maximum-Intensity Short-Term Voluntary Sustained Exercise in Man.

contributor:
MacDougall, J. D.
Kinesiology

subject: anaerobic energy exercise man

abstract: The purpose was to directly assess the relative contributions of the anaerobic energy releasing pathways to ATP provision during very brief (2-14s) maximal isometric contractions in human skeletal muscle, and to examine the recovery processes following such exercise. Eleven males performed unilateral sustained maximal isometric contractions of the knee-extensor muscles. Ten needle biopsy samples [2 pre-exercise (PreEx), 4 exercise (Ex), and 4 recovery (Rec) samples] were obtained from each subject over 3 separate testing sessions. Circulation was occluded at the upper thigh prior to all biopsies. Ex samples were taken immediately following 2-14s of maximal voluntary contraction. Contraction times were randomly pre-determined such that 6 biopsies were obtained following each of 2, 4, 6, 8, 10, 12, and 14s of contraction. Rec samples were collected 10, 20, 30, 40, 60, 120, and 180s following 14s maximum contractions. Significant changes occurred for both phosphocreatine (PCr) and lactate concentration within the initial 2s (n=7, p≤0.05) of contraction. In 14s, PCr concentration decreased to 53.3% of the PreEx value at a rate of 2. 53±0 .18 mmol· kg⁻¹dm · s⁻¹, while muscle lactate increased to approximately 8 times the initial value, at a steady rate of 1. 52±0 .10 mmol ·kg⁻¹ s⁻¹. Following contraction, PCr returned to the PreEx value within 60s of recovery (n=4, p>O. OS) , but muscle lactate concentration was still significantly elevated above resting after 180s of recovery (n=4, ≤0.05). Total ATP production over 14s was 69.03±4.47 ⁻¹dm, with an average turnover rate of 4.93±0.32 mmol·kg⁻¹dm·s⁻¹. The initial ATP turnover rate was 7. 26±1. 94 mmol· kg⁻¹ dm· s⁻¹ with 67% of the ATP being derived from PCr degradation and 33% from glycogenolysis. As contraction continued, however, there was an almost equal contribution to ATP provision from the 2 anaerobic energy delivery pathways, and (with the exception of the first 2 seconds) the proportion changed very little over time. This study was supported by the Natural Sciences and Engineering Research Council of Canada.

abstract: Thesis

abstract: Master of Science (MSc)


title: Biochemical Investigations on Adenosine Kinase and its Mutants

contributor:
Gupta, R. S.
Biochemistry

subject:
biochemistry
adenosine
adenosine kinase
mutants

abstract: In the present study, adenosine kinase (AK) was purified from a number of different sources (e.g. bovine liver, Syrian hamster liver, kidney and heart; Chinese hamster ovary cells and human placenta). The enzyme from bovine liver and Syrian hamster liver was purified to apparent homogeneity by a combination of ion-exchange chromatography, affinity chromatography and gel filtration chromatography. The purified enzyme showed a single band of 40 kDa in SDS-polyacrylamide gel electrophoresis, which was similar to that reported from other sources in the literature. A number of biochemical and enzymatic characteristics of AK were investigated. The reliability and reproducibility of the AK assay as established in previous studies was determined. The apparent Km of partially purified AK from Syrian hamster liver for adenosine was determined to be 0.16 uM, which is consistent with earlier reports. A novel result obtained in these studies is that AK activity was found to be completely dependent upon the presence of phosphate or other pentavalent ions. AK from different sources did not show any activity in the Tris-HCl buffer, pH 7.0, but the activity increased dramatically upon the addition of phosphate and it reached a maximum at 2 mM phosphate. There was no inhibition of AK activity when the phosphate concentration was increased up to 100 mM. AK could also be activated by substituting phosphate with either arsenate or vanadate, which have similar chemical structures to phosphate. The temperature inactivation kinetics of AK showed that AK from human fibroblast cells had higher thermal resistance than AK from Chinese hamster ovary cells at 50°C. The presence of phosphate had no effect on the thermal stability of AK. Antibodies to purified AK from Syrian hamster liver were raised in both rabbits and guinea pigs. Antiserum from the rabbit which gave the strongest response was used for further studies. AK was recognized by and reacted specifically with the antiserum at a dilution of up to 1: 16,000. The AK antibody which was covalently bound to Protein A Sepharose beads immunoprecipitated a 40 kDa protein from radio-labelled Chinese hamster ovary or baby hamster kidney cell extracts. In addition, this antibody preparation immunoprecipitated AK activity from Syrian hamster liver extracts. However, immunoblotting showed that all the antisera could not react with AK from Chinese hamster ovary cells and human cells, suggesting a strong species specificity. A partial protein sequence of AK was obtained by microsequencing of a cyanogen bromide fragment of purified AK from Syrian hamster liver. The sequence was Tyr-Val-Asp-Ser-Leu-Phe-Gly-Ala-Glu-Thr-Glu-Ala-Ala-Leu. Degenerate DNA probes for this sequence can now be made and used for either screening of eDNA libraries or carrying out PCR experiments to facilitate the cloning of the AK gene(s). Finally, conditions for selection of revertants from AK⁻ mutants of Chinese hamster ovary cells have been developed. The AK⁺ revertants from AK⁻ mutants can be obtained using adenosine, alanosine, uridine and erythro-9-(2-hydroxyl-3-nonyl) adenine in the growth medium. Three revertants have been isolated. These revertants regained their AK activities and lost their drug-resistance at same time. This method can also be used in the future to clone the AK gene by transfecting AK⁻ mutants with foreign DNA.

abstract: Thesis

abstract: Master of Science (MSc)


title: Effect of Coating Microstructure on the Electrochemical Properties of Continuous Galvanized Coatings on Press Hardened Steels

contributor:
McDermid, Joseph
Kish, Joseph
Materials Science and Engineering

subject:
Continuous Galvanizing
Zinc coatings
Press Hardened Steels
Electrochemical Properties

abstract:
In response to more stringent global CO2 emissions, automotive manufacturers have increased the use of advanced high strength steels (AHSS). Ultra-high strength steels are often used within the body-in-white (BIW) for safety critical parts and structural reinforcements, such as roof rails and side impact beams. Currently, the most commonly used press hardened steel (PHS) grade for these applications is 22MnB5, with a typical composition of 0.22C 1.2Mn 0.25Si 0.005B (wt%). Automotive OEMs have expressed a desire to use Zn-based coatings as they are compatible with the current painting system and have the potential to provide robust cathodic protection. The steel blanks generally undergo direct hot press forming (DHPF) to achieve the necessary martensitic microstructure and target mechanical properties, but this presents challenges for Zn-coated 22MnB5. The adoption of Zn-based coatings within the automotive industry has been inhibited by the prospect of liquid metal embrittlement (LME) resulting from DHPF, as well as the desire to provide robust cathodic protection.
	Previous literature has reported that a zinc ferrite (α-Fe(Zn)) coating with a global Zn content of at least 30 wt% will provide cathodic protection to the underlying substrate. The main goal of this work was to determine the microstructural evolution and electrochemical properties of galvanized (GI70 – 70 g/m2/side) 22MnB5 substrates as a function of the annealing time at a typical austenization temperature of 900°C. It was found that the Zn-based coatings annealed at 700°C consisted to a mixture of small volume fraction of α-Fe(Zn) and Г-Fe3Zn10. After heating to 900°C, the coating comprised varying volume fractions of α-Fe(Zn) and Zn(Fe) liquid, which transformed to Г-Fe3Zn10 after solidification. The relative fraction of Г Fe3Zn10 was found to decrease with increasing annealing time until the coating completely transformed to α-Fe(Zn) after annealing at 900°C for 240 s. GDOES results found that, when the sample was annealed at 900°C for 240 s, the global Zn content of the coating was less than 30 wt%. Coatings comprising varying fractions of Г-Fe3Zn10 were subjected to uniaxial tensile tests to determine how the coating microstructure affected the mechanical properties in comparison to the uncoated substrate material. It was found that the uncoated substrate material met the mechanical property requirements of σ(UTS)min ≥ 1500 MPa regardless of annealing time. However, σ(UTS) was found to decrease with increasing annealing times for the GI70 coated samples until the target mechanical properties were not met when the sample was annealed at 900°C for 180 s. This was attributed to increased coating thicknesses leading to a decrease in the martensitic cross-sectional area to support the load.
	Furthermore, the coatings were subjected to a variety of electrochemical characterization techniques, including potentiodynamic and galvanostatic polarization scans, potentiostatic scans, and electrochemical noise tests. Potentiodynamic polarization scans indicated a higher driving force for cathodic protection when the coating contained some fraction of Г-Fe3Zn10. Furthermore, a limiting current density for these samples was observed, demonstrating that Г-Fe3Zn10 corrodes at a slower rate in comparison to α Fe(Zn). Galvanostatic polarization measurements indicated that, when the fraction of Г Fe3Zn10 within the coating was below 15 vol%, the protective properties of the phase were not exhibited. XRD and TEM analysis revealed the formation of three corrosion products on the surface: simonkolleite, hydrozincite, and akaganeite. It was found that, when samples contained greater than 15 vol% Г-Fe3Zn10 in the coating, the predominant corrosion products were a combination of simonkolleite and hydrozincite. When the Г Fe3Zn10 content was below this value, the dominant corrosion product was found to be akaganeite. Furthermore, substrate attack was observed on a sample annealed at 900°C for 420 s when the coating layer was intact, indicating that the α-Fe(Zn) only containing coating obtained at this time does not provide cathodic protection.
	Based upon the current results, it was determined that a minimum volume fraction of 15 vol% Г-Fe3Zn10 must be present within the coating layer to obtain robust cathodic protection. Furthermore, it was determined that the processing window to develop cathodically protective Zn based coatings while mitigating LME is extremely narrow. This is a result of the fact that it is necessary for at least 15 vol% Г-Fe3Zn10 to be present within the coating microstructure at room temperature, which is liquid at the forming temperatures of 900°C. From the current findings, it was found that it is unlikely that a cathodically protective Zn-based coating can be obtained for DHPF steel parts using 22MnB5 as a substrate material. This is due to the high forming temperature resulting in liquefication of the coating and the rapid cooling rates necessary to achieve the target mechanical properties of σ(UTS)min ≥ 1500 MPa. Thus, it is recommended that the current substrate material be altered such that the part may be formed below the peritectic temperature of 782°C.

abstract: Thesis

abstract: Master of Applied Science (MASc)


title: Sequencing of Rabbit Brown Adipose Tissue Uncoupling Protein cDNA: Characterization of Rat and Rabbit Uncoupling Protein mRNAs

contributor:
Freeman, K. B.
Biochemistry

subject: rabbit rat protein cDNA mRNA adipose

abstract:
A cDNA clone encoding the entire amino acid sequence of rabbit brown adipose tissue uncoupling protein has been isolated and sequenced. The coding region of this cDNA is 80.6% identical to that of the rat uncoupling protein cDNA. In contrast to rat uncoupling protein for which there are two mRNAs of 1500 and 2000 nucleotides there is only one rabbit uncoupling protein mRNA of 2000 nucleotides. Whereas the rat cDNA hybridizes more strongly to the shorter rat uncoupling protein mRNA the rabbit cDNA hybridizes more strongly to the longer rat uncoupling protein mRNA. Primer extension and Northern blot analysis were performed to try to account for the difference of 430 ± 75 nucleotides between the two rat uncoupling protein mRNAs. Northern blot analysis indicated the presence of 355 more nucleotides in the 3'-untranslated region of the 2000 nucleotide long rat uncoupling protein mRNA than in the 1500 nucleotide long rat uncoupling protein mRNA. The two rat uncoupling protein mRNAs could therefore arise by differential processing. Primer extension revealed that the two rat uncoupling protein mRNAs have a 5'-untranslated region of approximately 186 nucleotides. The deduced amino acid sequence of rabbit UCP is 86% identical with both the rat and hamster proteins. Several regions are conserved in all three uncoupling proteins. The two longest regions of conservation are residues 52 to 69 and 82 to 100 of the mature proteins and correspond to two of several basic regions of the protein that have been suggested as possible targeting sequences. These conserved regions fall within amino acids 52 to 104 of the mature rat protein, which has been shown by others to target a passenger protein to mitochondria. Helical wheel diagrams that correspond to residues 52 to 68 and residues 72 to 92 reveal possible amphiphilic α-helical formations that may be involved in targeting. Regions corresponding to those conserved in the three UCPs are also conserved in three mammalian ADP/ATP carriers and may indicate a common role for these regions, perhaps including targeting. There is almost complete conservation of lysine, arginine, and cysteine residues that are thought to be involved in nucleotide binding and proton transport in the three UCPs.
There is a threonine to alanine change at the carboxyl-terminus of the rabbit protein compared to the rat protein. This amino acid difference may explain the differential reactivities of rabbit and rat UCP with an antibody preparation against rat UCP.

abstract: Thesis

abstract: Master of Science (MSc)


title: Active Site Studies on Microsomal Aminopeptidase

contributor:
Chan, W. W. -C
Biochemistry

subject:
microsomal
aminopeptidase
active site
active
porcine
pig

abstract: The active site of porcine kidney microsomal aminopeptidase was investigated using single, multiple and EDTA inactivation kinetic studies. Good inhibitors invariably contained a zinc-coordinating group such as the mercapto moiety, which proved to be the best ligand for aminopeptidase. Due to the potency of β -mercaptoethylamine, derivatives of this compound were examined for aminopeptidase inhibition. (S)-2-amino-4-methyl-l-pentanethiol (L-leucinthiol) exhibited the largest potency and specificity towards aminopeptidase when compared against carboxypeptidase A and thermolysin, two similar zinc-peptidases. The presence of a zinc-coordination subsite, two hydrophobic pocket subsites and a second amine-binding subsite (distinct from that responsible for substrate recognition) were discerned and the binding modes of amino acid hydroxamates and mercaptoamines compared using Yonetani-Theorell inhibition kinetics. Aminopeptidase does not show virtually any stereoselectivity between L-and D-leucine hydroxamate while greater than a 1,000-fold preference is seen for L-leucinthiol over the D isomer. Also, the amino group of mercaptoamines is crucial to the binding of these inhibitors whereas that of the hydroxamate compounds does not seen to contribute much to their binding. The differences in binding between hydroxamates and mercaptoamines are postulated to be a consequence of the product analogue nature of the former and transition state analogue character of the latter. L-leucine hydroxamate is proposed to bind in a backwards orientation while the D isomer binds in the normal substrate-like position. Similarly, L-leucinthiol is proposed to bind in the same fashion as substrate. Design of future inhibitors should endeavour to: (1) lower the pᵏₐ of the α-amino group, (2) include an extended chain structure capable of binding to additional hydrophobic pockets, (3) incorporate a second amine moiety into the structure to interact with the second amine-binding subsite and (4) replace the mercapto group with a more potent zinc ligand such as the selenol group.

abstract: Thesis

abstract: Master of Science (MSc)


title: mRNA Decapitation Induced by the Herpes Simplex Virus Virion Host Shutoff Protein

title: mRNA Decapitation Induced by VHS

contributor:
Smiley, James
Biology

subject:
mRNA
herpes simplex virus
VHS
protein

abstract: Cells infected with herpes simplex virus show a rapid cessation of protein synthesis and a dramatic decline in the levels of mRNA; a process known as host shutoff. This effect is attributed to a viral tegument protein called the virion host shutoff protein, or vhs. The mechanism by which vhs induces mRNA degradation is not yet understood. It is not known whether vhs possesses RNase activities or if it acts in combination with other cellular factors. To gain a better understanding of the function of vhs, I examined RNA degradation in detail by analyzing the RNA decay products generated in the presence of vhs. 𝘐𝘯 𝘷𝘪𝘷𝘰 experiments, performed by infecting murine erythroid leukemia cells with HSV, revealed that beta-globin mRNA is rapidly degraded, in the presence of vhs, without being converted to detectable decay intermediates. The half-life of this mRNA was 15 and 60 minutes for HSV-2 and HSV-1, respectively. Using vhs translated in a rabbit reticulocyte lysate system, I found that vhs induced rapid decay at the 5' end of a capped RNA molecule. The decay event was endonucleolytic and occurred at preferred sites downstream of the cap, generating capped oligonucleotides. Unlike influenza RNA polymerase, the cleavage event did not occur at a fixed distance from the cap since capped oligos of differing size were generated from different RNA substrates. My data indicate that vhs induced cleavage exhibits a strong, but not absolute preference for RNAs possessing an m⁷G cap, which may account for vhs' specificity for m RNAs 𝘪𝘯 𝘷𝘪𝘷𝘰.

abstract: Thesis

abstract: Master of Science (MSc)


title: Interactions Between Protein Kinase C and Arginine-Rich Peptides

contributor:
Epand, Richard
Biochemistry

subject: protein kinase arginine peptides

abstract: Protein kinase C (PKC) is translocated to a phospholipid bilayer by calcium. Once at the membrane protein kinase C undergoes a conformational change which results in the removal of the pseudosubstrate domain from the active site. The enzyme then phosphorylates Ser/Thr residues on positively charged substrates. Certain substrates, however, can undergo cofactor independent phosphorylation by producing a conformational change in the enzyme in the absence of phospholipid and calcium. Studying the conformational change in PKC by physical techniques is difficult to perform with a phospholipid bilayer present. To study the conformational change in PKC in the absence of a membrane, the interactions between an Arginine-rich peptide (ARP), which underwent cofactor independent phosphorylation, and PKC was investigated. The Kₘ and kcₐₜ of the enzyme for ARP, in the absence of cofactors, was around 10 μM and 0.38 s⁻¹, respectively. The Kₘ did not significantly change upon the addition of phosphoipid and calcium. However, the kcₐₜ increased 2-3 fold in the presence of phospholipid and calcium. In the absence of phospholipid and calcium, ARP induced the exposure of hydrophobic site(s) on the enzyme. Additionally, ARP was able to promote the translocation of PKC to the membrane in the absence of calcium. PKC translocated to the membrane by ARP displayed the same susceptibility as the calcium membrane bound enzyme to limited proteolytic cleavage. Therefore, both ARP and calcium induce a similar membrane bound conformation in PKC. Additionally, the binding of ARP to PKC seems to occur through at least one high affinity site apart for the active site. These results demonstrate new insight into cofactor independent phosphorylation by PKC as well as illustrate a novel mechanism by which a substrate can promote the translocation of PKC in the absence of calcium.

abstract: Thesis

abstract: Master of Science (MSc)


title: Reactivation of UV-Irradiated Adenovirus Type 2 and Herpes Simplex Virus Type 1 in Mammalian Cells

title: Reactivation of UV-Irradiated AD 2 and HSV-I in Mammalian Cells

contributor:
Rainbow, Andrew
Health and Radiation Physics

subject:
UV radiation
adenovirus type 2
herpes simplex type 1
mammal
cancer

abstract: Much research is being conducted into the causes of human cancer. A number of human autosomal recessive diseases such as Xeroderma Pigmentosum are characterized at least in part by a defect or aberration in one or more forms of DNA repair and at the same time an elevated incidence of cancer. Also, carcinogens cause mutations in DNA and the greater the carcinogenicity, the greater the mutagenicity. As a result, much attention has been focused on DNA repair and its relationship to cancer incidence. The HCR of V antigen formation by UV-irradiated Adenovirus type 2 (Ad 2) was examined using apparently normal human fibroblasts, tumor cells (HeLa CCL2), and cells transformed by Ad 5 DNA (293, 293 N3S). A decrease in the HCR of V antigen formation was found for HeLa CCL2 cells as compared to apparently normal human fibroblasts, but not for the transformed cells. These results are discussed in terms of the characteristics of the cell types. Herpes simplex virus type I encodes a polymerase and thymidine kinase (tk) activity which are involved in viral DNA synthesis. Paa	᷇ 5, an HSV-1 mutant containing one or more mutations in the polymerase gene is an antimutator. If these are also involved in viral DNA repair, then the HSV-1 polymerase, tk activity, and mutant polymerases conferring altered mutation rates should provide excellent tools with which to probe cellular DNA repair processes and mutagenesis. The study of the HCR of plaque forming ability of HSV-1 KOS wild type (WT), Paa	᷇ 5 and PTK3B (lacking thymidine kinase activity ) using VERO cells revealed a decrease in the HCR of Paa	᷇  5 and increase of surviving fractions of PTK3B with respect to that of HSV-1 KOS WT. Similar studies using apparently normal human fibroblasts, Xeroderma Pigmentosum and Cockayne Syndrome cells also implicated the HSV-1 polymerase in viral DNA repair. The results are discussed in terms of the function of the HSV-1 polymerase and the DNA repair abilities of XP and CS cells.

abstract: Thesis

abstract: Master of Science (MSc)


title: DNA Microarray Images: Processing, Modelling, Compression

contributor:
Shirani, S.
Deen, M. J.
Electrical and Computer Engineering

subject:
DNA
images
processing
modelling
electrical engineering

abstract: DNA Microarray is an innovative tool for gene studies in biomedical research. It is capable of testing and extracting the expression of large number of genes in parallel. Its applications can vary from cancer diagnosis to human identification. A DNA microarray experiment generates an image which has the genetic data embedded in it. Fast, accurate, and automatic routines for processing and compression of these images do not exist. For processing and modelling of micoarray images, we introduce a new, fast and accurate approach in this thesis. A new lossless compression method for microarray images is introduced that provides an average compression ratio of 1.89:1, and that outperforms other lossless compression schemes and the work of other researchers in this field. For the lossy compression, our new method has overcome the rate-distortion curve of JPEG. A new scanning method called spiral path, and a new spatial transform called C2S are introduced in this thesis for lossless and lossy compression of microarray images.

abstract: Thesis

abstract: Master of Applied Science (MASc)


title: The Effect of Creatine Supplementation on Body Composition, Intramuscular Phosphates and High Intensity Exercise Performance in Males and Females

title: Creatine and Performance in Males and Females

contributor:
Tarnopolsky, Mark
Human Biodynamics

subject: male female exercise creatine

abstract: Background and Rationale: Creatine phosphate (PCr) is degraded 𝘷𝘪𝘢 the creatine kinase (CK) enzyme to provide energy to rephosphorylate ADP back to ATP during high-intensity muscle contractions. We, and others, have previously shown that dietary creatine (Cr) loading can improve performance in short-duration, high intensity exercise in males and females. An increase in fat-free mass has also been reported, which was higher for males compared to females. It was unknown whether dietary Cr loading increases intramuscular [Cr] equally in both genders. The purpose of this investigation was to examine the effect of oral Cr loading upon [PCr] and [Cr] and exercise performance in both males and females. Methods: Twenty-seven healthy young subjects (n = 13 male, n = 14 female) participated in the investigation. Subjects performed an ischemic handgrip test, maximum voluntary contraction of dorsiflexors and knee extensors, a 30 s maximal cycling test (Wingate), and body composition (by DEXA scanning) was determined before and after supplementation with either Cr (n = 13) (5 g by mouth 4 •d⁻¹ x 5 d, followed by 5 g•d⁻¹ x 5 d) or placebo (pl; n = 14). Muscle biopsy samples were obtained before and after supplementation and were assayed for high-energy phosphates ([Cr], [PCr] and [ATP]). Results: Cr supplementation resulted in an increase in intramuscular total creatine concentration [TCr] (pl, 129.9 ± 13.8 to 132.9 ± 10.8 mMol•kg⁻¹ dw; Cr, 129.9 ± 11.6 to 146.4 ± 20.1 mMol•kg⁻¹ dw, p < 0.05) and a difference in [PCr] (pl, 72.8 ± 15.2 to 67.3 ± 4.3 mMol•kg⁻¹ dw; Cr, 70.9 ± 7.4 to 77.1 ± 13.2 mMol•kg⁻¹ dw), but had no effect on [Cr], and no differences in gender were observed. DEXA analysis showed no significant increase in total or fat-free mass; similarly, no improvements in maximum voluntary contractions were observed in either gender. In Wingate testing, males had a significant increase in peak power (pl, 971 ± 72 W to 989 ±76 W versus Cr, 994 ± 174 W to 1042 ± 154 W) and peak power per kilogram of body weight (pl, 12.8 ± 1.3 to 12.9 ± 1.1 W•kg⁻¹; Cr, 11.5 ± 1.1 to 12.1 ± 1.3 W•kg⁻¹) as a result of creatine supplementation, while females showed no significant difference. Conclusion: Creatine monohydrate supplementation at 20 g•d⁻¹ for 5 days resulted in an increase in muscle total creatine concentration for males and females, but did not affect body composition or maximum voluntary contraction. Males showed improved performance in absolute and relative peak power generation with supplementation while females did not. This may suggest that males and females respond differently to Cr supplementation.

abstract: Thesis

abstract: Master of Science (MSc)


title: Maximum Likelihood Star Alignment of Multiple Molecular Sequences

contributor:
Jiang, Tao
Electrical and Computer Engineering

subject: star alignment molecular

abstract: In the study of pairwise sequence alignment, a clear relationship between the scoring system and assumptions about the occurrence of evolutionary events has been established in [BT86], [TKF91], [TKF92] and [TC95] by proposing an evolutionary model. To align two given sequences, one need estimate some evolutionary parameters through maximum likelihood method, and find an alignment with the maximum probability using the estimated parameters. In this thesis, we extend the above model and the maximum likelihood method to star alignment of three molecular sequences along the same line. We overcome the duplications of star alignments by defining canonical star alignments. Two star alignment algorithms, i.e. sum approach and direct alignment approach, are proposed in this thesis based on two different likelihood functions. A software system, called MLSAS (Maximum Likelihood Star Alignment System), is developed to implement the two algorithms with a friendly graphical user interface. Simulation studies show their behaviors are satisfactory for closely related sequences. A few real examples are also provided.

abstract: Thesis

abstract: Master of Engineering (ME)


title: Effects of Short-Term Creatine Supplementation on Whole-Body Protein Metabolism

subject: creatine protein metabolism

abstract: Creatine-monohydrate (CrM) supplementation has been shown to increase body mass and fat-free mass (FFM), however, the mechanism by which CrM affects body composition has not been determined. We investigated the effects of short-term CrM supplementation on whole-body protein turnover in 27 recreationally active male and female volunteers. Subjects underwent measurements prior to and following 9-10 days of CrM (20 g/d x 5 d, 5 g/d x 4-5 d) (n =14), or placebo (PL) (n =13) supplementation. Protein turnover was assessed using L-[1-¹³C] leucine stable isotope tracer, urinary urea nitrogen (N) excretion, and N-balance (Nbal) techniques. Total body mass (TBM), leucine flux, leucine oxidation, non-oxidative leucine disposal (NOLD), 24-hr urinary urea N excretion, and Nbal were determined before and after treatment. Additionally, the effects of CrM supplementation on renal function and metabolite clearance were evaluated by measuring creatinine (CTN) excretion, plasma CTN concentration, and CTN clearance. There was no effect for CrM as compared to PL on TBM, leucine flux, leucine oxidation, or NOLD. However, leucine oxidation was lower for the CrM-treated males as compared to the PL-treated males following supplementation (P < 0.05). Leucine flux and NOLD were higher for the males vs. the females (P < 0.05). Neither urinary urea N excretion nor Nbal were affected by treatment. Plasma [CTN], CTN excretion, and CTN clearance were also unchanged for CrM vs. PL. These findings suggested that CrM supplementation may have an effect upon leucine oxidation in males, yet there were no effects seen in females, nor were other indices of leucine turnover altered by CrM supplementation. Furthermore, short-term CrM supplementation did not have any adverse effects on renal function.

abstract: Thesis

abstract: Master of Science (MSc)


title: Effects of Acute Creatine Supplementation on Resting Muscle Protein Fractional Synthetic Rate

subject: creatine muscle protein synthetic

abstract: Background & Rationale: During high intensity muscle contractions phosphocreatine is enzymatically degraded by creatine kinase (CK) to form creatine and the free energy which is released used to phosphorylate ADP to ATP. Creatine is then rephosphorylated during periods of relative ATP abundance (ie. rest) by CK back to phosphocreatine. Recognition of the importance of the phosphocreatine system to energy transduction has led many to believe that creatine monohydrate supplementation, which may lead to increases in phosptocreatine (PCr), may be beneficial during high intensity exercise. Several studies have demonstrated that creatine monohydrate supplementation for as few as three days can result in significant performance gains during exercise such as sprinting, or a weight lifting program. A common observation during these studies is a 1-2 kg increase in lean body mass (LBM). Although most researchers have speculated that this increase in LBM is due to water retention, some 𝘪𝘯 𝘷𝘪𝘵𝘳𝘰 work has demonstrated that creatine may stimulate protein synthesis. The purpose of this investigation was to examine whether a loading dose of creatine (20g/d x 7d) would have any affect on mixed muscle protein synthesis (MPS) in resting human skeletal muscle. Methods: A total of 22 young healthy subjects (n = 11 male, n = 11 female) were included in the study. On the day of measurement, subjects were provided with a meat free pre-packaged diet that was based upon individual diet records. Measurements of mixed muscle protein fractional synthetic rate (FSR) were completed over a 14 h resting period using a primed constant infusion of L[1-¹³C]leucine and muscle biopsies of 𝘲𝘶𝘢𝘥𝘳𝘪𝘤𝘦𝘱𝘴 𝘧𝘦𝘮𝘰𝘳𝘪𝘴 at isotopic plateau. Subjects were then randomly assigned to either a creatine (20g/d x 7d) or a placebo (isoenergetic glucose polymer) group. Following 7 days of supplementation, subjects reported to the lab under the same conditions as in the pre-trial, and resting mixed muscle protein FSR was again determined. Results: There were no significant between group differences in the baseline subject characteristics. No significant difference in FSR was observed with regards to condition (Pl: pre-0.63 ± 0.02 %/h; pst-0.71 ± 0.016 %/h; Cr: pre-0.56 ± 0.02 %/h; pst-0.58 ± 0.023 %/h) (creatine supplementation), time, or gender (Males: pre-0.06 ± 0.02 %/h; pst-0.068 ± 0.023 %/h; Females: pre-0.057 ± 0.02 %/h; pst-0.058 ± 0.015 %/h) Creatine supplementation resulted in a 13.1% increase in total creatine, however, no significant increases; in PCr or free Cr were observed. Similarly, no significant increases for fat free mass (FFM), or total mass were observed. Conclusion: It is concluded that creatine monohydrate supplementation for 7 d at 20g/d significantly increases muscle total creatine concentration, however, does not significantly affect muscle protein FSR in males or females.

abstract: Thesis

abstract: Master of Science (MSc)


title: Biosynthesis, Resistance and Resistance Regulation of the Glycopeptide Antibiotic A47934 in Streptomyces toyocaensis NRRL 15009

title: Biosynthesis, Resistance and Regulation of the Glycopeptide Antibiotic A47934

contributor:
Wright, G. D.
Biochemistry

subject: antibiotic glycopeptide resistance regulation

abstract: Multiple antibiotic resistant bacteria continue to be a threat to the health of the world's population. Glycopeptide antibiotics are one type of drug that are used to treat these serious pathogens. Increased usage over the years has led to the emergence of bacteria which are resistant to these glycopeptide antibiotics and now the need for altered antibiotics with an increased effectiveness has arisen. 𝘚𝘵𝘳𝘦𝘱𝘵𝘰𝘮𝘺𝘤𝘦𝘴 𝘵𝘰𝘺𝘰𝘤𝘢𝘦𝘯𝘴𝘪𝘴 NRRL 15009 produces the glycopeptide antibiotic A47934. Here, the biosynthetic gene cluster for A47934 was sequenced in its entirety. All enzymes encoded by assigned open reading frames were analyzed and functions assigned where possible. The resulting biosynthesis cluster encodes all the enzymes necessary to produce A47934, as well as confer resistance and regulate the resistance response. In addition to sequencing the biosynthetic gene cluster, enzymatic studies were attempted on the two-component regulatory system (VanR and VanS) which confers resistance to A47934. Finally, inactivation of 𝘴𝘵𝘢𝘓 was attempted. Overall, the results presented here should help us to further understand how these chemically complex glycopeptide antibiotics are made and lend further insight into how we can attempt to produce new semi-synthetic versions.

abstract: Thesis

abstract: Master of Science (MSc)


title: Ash Density and Trabecular Bone Structure in Proximal Femur Fracture

contributor:
Webber, C. E.
Medical Physics

subject: bone density femur fracture

abstract: The likelihood of a given bone fracturing atraumatically is difficult to assess.. A number of factors - age, frequency of falling, cushioning by overlying tissues, protective reactions - as well as bone strength determine fracture risk. The interrelationships between these factors make it hard to evaluate the effect of any one variable in a study. The most common method of assessing bone strength is to use the surrogate method of bone mineral content (BMC) or bone mineral density (BMD). The architectural breakdown of the trabeculae, the mineral "mesh" which makes up bone, is a little-studied factor which may help to better predict fracture. In this thesis, the results of quantitative measurements of trabecular architecture, BMD, and strength of femoral heads scavenged from hip replacement surgery will be presented. This is intended to illuminate the relationships between bone strength, bone density, and trabecular architecture.

abstract: Thesis

abstract: Master of Science (MSc)


title: The Role of Liver-X-Receptor and Retinoid-X-Receptor in the Regulation of Tumour Necrosis Factor-a Expression and Production in Human Monocytes

title: Regulation of TNF-a in Monocytes by LXR and RXR

contributor:
Capone, J. P.
Biochemistry

subject:
liver
retinoid
receptor
tumour necrosis
monocytes

abstract: Liver X receptor (LXR) is a member of the nuclear hormone receptor superfamily that is activated by hydroxylated cholesterol derivatives referred to as oxysterols. It has also been shown to play a crucial role in regulating cholesterol trafficking and lipid metabolism in liver and macrophages. Furthermore, LXR. has also been directly implicated in the reduction of atherosclerosis in several murine models of the disease by virtue of its ability to promote reverse cholesterol efllux from intima-resident lipid-loaded macrophages. While roles for LXR in monocyte biology have focused primarily on cholesterol trafficking, evidence for other functions for the receptor outside of its traditional role as a mediator of cholesterol homeostasis is lacking. Presented herein is evidence that LXR also serves as a mediator of cytokine expression. This work has shown that treatment of human peripheral blood monocytes or monocytic THP-1 cells with the LXR ligand 22(R)-hydroxycholesterol (22R-HC), in combination with 9-cis-retinoic acid (9cRA), a ligand for the LXR. heterodimerization partner retinoid X receptor (RXR), results in the specific induction of the potent pro-apoptotic and pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-α.). Promoter analysis, inhibitor studies, and order-of-addition experiments demonstrated that TNF-α. induction by 22R-HC and 9cRA occurs by a novel two-step process. The initial step involves 22R-HC-dependent induction of TNF-α. mRNA, and intracellular accumulation of TNF-alpha protein, mediated by binding of LXRα/RXRα to an LXR response element at position -879 of the TNF-α promoter. Subsequent cell release of TNF-alpha protein occurs via a separable RXR-dependent step that requires de novo transcription and protein synthesis. Furthermore, the RXR-dependent secretory event can be mimicked by agents that induce monocytic differentiation like phorbol esters that culminate in RXR activation by a pathway that does not require exogenous ligand. In this context, RXR was also shown to be a down stream target of the protein kinase C (PKC) signal transduction cascade, that results in the activation of RXR and the induction of secretory factor(s) which facilitate secretion of LXR-derived TNF-α. These studies have provided evidence that should help to expand the currently known role for LXR in monocyte biology and have furthermore identified a new role for RXRs in promoting the secretion of soluble factors like cytokines. Furthermore, in light of reports that show LXR activity promotes a reduction in atherosclerosis, it stands to reason that this regulatory circuit of LXR-dependent production of TNF -α from monocytes would similarly contribute to the attenuation of atherosclerosis 𝘪𝘯 𝘷𝘪𝘷𝘰.

abstract: Thesis

abstract: Master of Science (MSc)


title: Local Deformation of the Erythrocyte Membrane Induced by Focused Light Illumination

contributor:
Fradin, Cecile
Physics and Astronomy

subject: erythrocyte light deformation illumination

abstract: A rapid local deformation of the erythrocyte membrane in the shape of an imprint caused by illumination with a focused laser beam in the presence of an external fluorophore has been investigated. This morphological change of the membrane appeared to be the very first observable step of the photohemolysis process which is exploited in photodynamic therapy. I showed that when a laser beam was focused on the erythrocyte membrane, the membrane was pulled toward the inside of the cell, independently of the direction in which the light was traveling. Imprint formation was observed neither on the lipid membrane of giant unilamellar vesicles nor on the membrane of nucleated mammalian cells such as HeLa cells. It shows that the effect is specific to erythrocytes; suggesting that it might be due to the unique structure of the erythrocyte membrane cytoskeleton. Also, I found that the rate of the imprint formation depended on laser input power, fluorophore concentration, and the presence of oxygen scavenger, but that it was independent of pH in the range pH= 6.4 to 7.8. These dependences are exactly the same for photohemolysis and for the imprint formation. By analogy with the photohemolysis process, these observations suggest that the imprint is created via the oxidization of band 3 proteins on the erythrocyte membrane. It supports the hypothesis that band 3 proteins play a pivotal role in the preservation of the erythrocyte shape. Preliminary work also suggests that spectrin is involved in the imprint formation process.

abstract: Thesis

abstract: Master of Science (MSc)


title: Interspecies Exchange Mutagenesis of the First Epidermal Growth Factor-Like Domain of Coagulation Factor VII

title: Interspecies Exchange Mutagenesis of the FVII EGF-1 Domain

contributor:
Clarke, Bryan
Biology

subject: mutagenesis epidermus growth coagulation interspecies

abstract: A high degreee of structural and sequence homology exists between the EGF -1 domains of the vitamin K-dependent coagulation factors, as well as between the EGF-1 domains of individual vitamin K-dependent coagulation factors from various species. Through studies of protein evolution it has been determined the conserved amino acid residues observed are essential for protein structure while the variable residues have been implicated in specific protein-protein interactions. In the case of FVII, this interaction is the high affinity binding of its cofactor, TF, which initiates the extrinsic pathway of coagulation. 43% of the contact area of the FVII molecule in the FVIIa•TF complex is located within the FVII EGF-1 domain. A series of human FVII variants have been constructed in which the EGF-1 domain has been exchanged, either in its entirety or as single amino acid substitutions, with that of the mouse or rabbit. These species were chosen as it had previously been shown that plasma from mouse or rabbit, when combined with human TF, was able to clot at a significantly greater rate than homologous human plasma. We hypothesized that through these exchanges it might be possible to generate a human FVII variant with increased TF binding. Of the FVII variants generated, 2 human FVII point mutants have shown an increased affinity for human TF after transient expression. A75D and T83K exhibited TF binding at 200% and 150%, respectively, of that seen with wild-type human FVII. Both A75D and T83K exhibited clotting and amidolytic activity that was proportionally increased, with respect to TF binding. Computer-generated structures of these variants predict an additional hydrogen bond between the FVII and TF molecules likely to be responsible for the increased TF affinity in the T83K mutant. Intramolecular forces within the FVII molecule are predicted to have caused a conformational change with the A75D mutation which has lead to its increased TF binding. The greater affinity for TF exhibited by the human FVII point mutants A75D and T83K, as compared to wild-type human FVII, is an important step toward the creation of new class of competitive inhibitors of coagulation which are specifically directed towards its initial stage, rather than later in the cascade. However, more study is needed to determine the ability of either of these mutants to compete against each other for TF both 𝘪𝘯 𝘷𝘪𝘵𝘳𝘰 and 𝘪𝘯 𝘷𝘪𝘷𝘰. If either mutant is successful, it can then be active site inhibited by site-directed mutagenesis or through the use of chloromethyl ketones, in order to decrease its clotting and amidolytic activity.

abstract: Thesis

abstract: Master of Science (MSc)


title: Investigation of Red Blood Cell/Foreign Surface Contact: The Deposition of Membrane Derived Material

title: Red Cell Membrane Components Deposited on Contacting Surfaces

contributor:
Brash, J. L.
Chemical Engineering

subject:
red blood cell
foreign surface
membrane
deposition

abstract: An investigation of red blood cell interactions with contacting foreign surfaces is reported. The purpose of the study was to provide evidence to test the hypothesis that red cell membrane related deposit occurs when a suspension of red cells are exposed to a foreign surface. This membrane derived deposit is speculated to inhibit fibrinogen and albumin adsorption to glass and polyethylene (1,2). Experiments consisted of flowing suspensions of washed human red cells through a column packed with spherical glass beads. The column is then washed to remove suspended cells and eluted using a mild detergent solution. The red cells were observed to have retained their biconcave shape after passage through the column. The eluates are examined by UV-visible spectroscopy and SDS-PAGE, and are found to contain cell membrane components. It is therefore concluded that membrane material had been deposited on the bead surface. The SDS-PAGE data show that membrane skeleton proteins are missing from the eluate, while SEM examination indicates the presence of filamentous deposits on the bead surfaces. These data suggest that cell-surface interactions may occur through a tether-type mechanism involving extrusion of part of the red cell membrane. A 3 level, 3 factor fractional factorial design was used to quantitatively investigate the deposition in the column and to assess the effect of experimental variables on the phenomenon. The three factors studied were hematocrit, surface shear rate, and the presence of plasma in the suspending medium. Statistical analysis of the data generated indicates a positive correlation between hematocrit and the amount of surface deposit. However no correlation for the effects of shear rate or plasma content could be established In summary, red cell membrane components on contacting foreign surfaces were identified and a preliminary description of the phenomenon involved obtained. The deposit of membrane material is discussed in relation to Uniyal's "red cell effect" (1), and in the context of foreign surface induced thrombosis. It is hoped that this work will promote greater respect for the red cell as an important element in blood biomaterials interactions, and will prompt continued investigation on Uniyal's and other potential "red cell effects".

abstract: Thesis

abstract: Master of Engineering (ME)


title: Gymnastic Training and Bone Mass in Prepubescent Females: Magnitude and Volume Effects of Impact Loading

title: Gymnastic Training and Bone Mess in Prepubescent Females

contributor:
Blimkie, C. J. R.
Human Biodynamics

subject:
gymnastics
bone
female
prepubescent
magnitude
impact loading

abstract:
Nineteen elite (E) gymnasts (>15hours/week gymnastic training), 14 high recreation (HR) gymnasts (8-15hours/week), and 15 low recreation (LR) gymnasts (1-7. 9 hours/week) were investigated to determine the effects of varying volumes of gymnastic training on bone mineral density (BMD) in prepubescent girls. Two normoactive control groups were additionally investigated to determine whether there was a magnitude effect of mechanical loading on BMD: 16 controls (C) and 15 height-and weight-matched controls (M). The mother of each daughter was measured in order to control and investigate the familial component of bone mass. Areal bone mineral density at the left proximal femur, lumbar spine (LS), and whole body (WB) and % body fat were measured by dual energy x-ray absorptiometry (DXA), and volumetric BMD was measured at the distal radius by peripheral QCT (pQCT). DXA BMD measures were corrected for bone size and expressed as bone mineral apparent density (BMAD). The HR group was significantly younger (8.68 ± 0.844 y, mean± SD) than both the E (10.02 ± 0.776 y) and C (9.96 ± 0.898 y) groups. The C group was significantly heavier (38.88 ± 4.868 kg) than the E (27.15 ± 2.819 kg), HR. (25.44 ± 3.564 kg), LR (32.98 ± 5.786 kg), and M (26.95 ± 3.301 kg) groups. Additionally, the LR group was significantly heavier than all other groups, with the exception of the C group. Femoral neck (FN) BMD was only significantly different between the E (0.706 ± 0.051 g•cm⁻²) and LR (0.649 ± 0.069 g•cm⁻²) groups. FNBMAD was only greater in the E (0.232 ± 0.048 g•cm⁻³) group compared to the C (0.191 ± 0.052 g•cm⁻³) group. LSBMAD and WBBMAD were significantly greater in both E (0.233 ± 0.019 and 0.100 ± 0.008 g•cm⁻³) and HR (0.239 ±
0.038 and 0.100 ± 0.006 g•cm⁻³) groups when compared to the LR (0.212 ± 0.022 and
0.090 ± 0.008 g•cm⁻³) and C (0.219 ± 0.020 and 0.085 ± 0.004 g•cm⁻³) groups, respectively. Total radial and cortical radial BMD was greater in both E (360.50 ± 51.569 and 484.28 ± 70.179 mg•cm⁻³) and HR (373.10 ± 45.318 and 480.66 ± 46.720 mg•cm⁻³) groups compared to the C (296.61 ± 29.677 and 426.144 ± 37.652 mg•cm⁻³) and M
(306.42 ± 24.430 and 414.571 ± 25.194 mg•cm⁻³) groups, respectively. Radial trabecular BMD was greater in both E (211. 19 ± 38. 202 mg•cm⁻³) and HR (212. 61 ± 44.299 mg•cm⁻³) groups compared to the LR (175.89 ± 29.191 mg•cm⁻³), C (162.68 ± 27.304 mg•cm⁻³), and M (171.05 ± 30.639 mg•cm⁻³) groups. There were no significant differences for any bone measure among the groups of mothers. Mother-daughter correlations were relatively weak, and often insignificant, for BMD measures (r = 0.10-0.37), but strong for radial morphometric measures (r = 0.43-0.55). Radial trabecular BMD (r = 0.37; p<0.01) was more significantly correlated with gymnastic training volume (hours/week) than radial cortical BMD (0.30; p<0.05). These results suggest that there is a volume of training effect on BMD and a magnitude effect of mechanical loading on BMD. It appears that trabecular bone at the distal radius may adapt more rapidly or be more sensitive than cortical BMD to the strains imposed by impact loading. Additionally, it appears that, during prepubescence in females, bone morphometric properties may be more genetically regulated than bone mineralization.

abstract: Thesis

abstract: Master of Science (MSc)


title: Immune System Response to Changes in Training Intensity and Volume in Runners

contributor:
MacDougall, J. D.
Human Biodynamics

subject:
immune system
training
run

abstract:
This study examined the acute and chronic effects of changes in training volume and intensity on the blood lymphocyte percentages and immunoglobulin levels in runners. Twelve runners participated in four 10-day phases over a 40 day training period. Phase 1 and phase 3 were "baseline" phases of low volume/low intensity running (LV/LI). Phase 2 and phase 4 consisted of either high volume/low intensity (HV/LI) or high volume/high intensity (HV/HI) training. Subjects were randomly assigned to one of 2 different crossover training group orders: 1) LV/LI, HV/LI, LV/LI, HV/HI or 2) LV/LI, HV/HI, LV/LI, HV/LI. HV/LI training involved
doubling the LV distance run at the same LI of 60-70% V02 max. The HV/HI phase consisted of the same volume as the HV/LI phase, but alternate days were replaced with a series of high intensity 1000 m intervals (95-100% V02 max). Venous blood samples were drawn at rest on days 1, 4 and 7 and 5 minutes post-exercise on days 1 and 7 of each 10-day phase. Lymphocyte subsets were determined by flow cytometry using monoclonal antibodies for total T (CD3+), T-helper (CD4+), T-suppressor (CDS+) lymphocytes and HLA-DR+ (a B cell and "activated" T-lymphoid cell marker). IgA, IgG and IgM levels were obtained by ELISA analysis. This study revealed remarkable stability of humoral (antibody) immune components during and after runs of various intensities and volumes. Immunoglobulin levels were not significantly affected by alterations in volume or intensity over the 4 training phases. A transient decrease was observed in the T-lymphocyte percentages of CD3+, CD4+ and the CD4/CD8 ratio 5 minutes post-exercise which was significant (p <.05) during the HV/LI and HV/HI phases. Adaptation of lymphocyte subpopulations occurred with repeated exposure to increases in volume and intensity. A training order effect was suggested whereby an initial HI phase, was more immunosuppressive and possibly negated the effects of subsequent HV phases. Results indicate that the exercise-induced lymphocyte subset reduction is transient and suggest that the extent of the reduction is more dependent upon training intensity than volume, and the order of exposure to the high-intensity stimulus may determine the magnitude of subsequent responses.

abstract: Thesis

abstract: Master of Science (MSc)


title: Study of a New Source for Dual Proton Absorptiometry of the Lumbar Spine

subject:
photon
absorptiometry
lumbar spine
spine
lumbar

abstract: Bone disease and metabolic bone disorders are characterized by decrease in bone mineral content (BMC) and the vertebrae are primarily affected in osteoporosis. Thus it is essential to monitor changes in BMC due to disease, growth or therapy. Dual photon absorptiometry (DPA) is as yet the most efficient method for BMC determination in the lumbar vertebrae; most previous investigators have used ¹⁵³Gd which is generally accepted as possessing the ideal dual photon energy combination. Davis and Webber (1978) developed a method for routine production of ¹⁵³Sm which emits photons of nearly the same energy as ¹⁵³Gd and suggested its use in DPA. In the present study using ¹⁵³Sm, measurements with aluminum standards immersed in water resulted in errors of 4% and 4.5% in accuracy and precision respectively for mass of Al in the range 0.4-1.8 g.cm⁻². These results were obtained using different volumes of water, verifying that bone mineral mass determined by this method is independent of soft tissue content. Experiments with bone mineral phantoms gave values which correlated highly (r=0.97) with results obtained on a commercially available clinical densitometer using ¹²⁵I. Considering that these results are comparable with those obtained earlier using ¹⁵³Gd, that ¹⁵³Gd is scarcely available and several hundred times the cost, ¹⁵³Sm promises to be the potential source for routine clinical measurement of osteopenia in the lumbar spine by DPA. However, further invitro and invivo studies are necessary before it can be used regularly in nuclear medicine departments.

abstract: Thesis

abstract: Master of Engineering (ME)


title: The Behaviour of Fibrinogen at Artificial Surfaces

title: Fibrinogen at Artificial Surfaces

subject:
fibrinogen
artificial surfaces
artificial
behaviour

abstract: The adsorption of fibrinogen from human plasma onto various surfaces was investigated under differing conditions of plasma composition. Previous studies by Vroman have indicated that fibrinogen is only transiently adsorbed from normal human blood plasma, possibly due to displacement by high molecular weight kininogen (HMWK). It is possible that this "Vroman Effect" is a manifestation of the activation of the intrinsic clotting pathway responsible, in part, for thrombus formation on artificial surfaces. The effect was studied using dilutions of human plasma from 0.01% to 20.0% of normal concentration trace-labelled with a small amount of ¹²⁵I labelled fibrinogen. Adsorption of fibrinogen to various materials, including glass and several potentially useful biomaterials was measured. Fibrinogen is initially adsorbed apparently under diffusion limitation and later displaced at a rate which depends on the type of material being tested. There does not appear to be a direct relationship between material properties (contact angle, biocompatibility) and the "Vroman Effect". Studies comparing the effect for a series of clotting factor deficient plasmas seem to indicate that plasminogen and HWNK are the main displacing agents on glass. Factors XI and XII, prekallikrein and other components do not appear to be involved. An attempt to mathematically model the effect was hampered by the lack of adequate models to describe even one-component protein adsorption. An isotherm equation was developed based on a theoretical model of protein exchange and spreading on the surface. The resulting parameter estimates based on data for different surfaces were well conditioned and may provide a good in vitro basis for comparing materials. It is hoped that the theoretical model will also be compatible with dynamic adsorption and ultimately a multicomponent system such as plasma (and the Vroman Effect). This work may lead to a better understanding of blood-material interactions and may provide the basis for a simple in vitro test for the characterization of potential biomaterials.

abstract: Thesis

abstract: Master of Engineering (ME)


title: Structural Analysis of Aminoglycoside Modifying Enzymes: Towards Rational Drug Design

contributor:
Berghuis, Albert
Biochemistry

subject: aminoglycoside enzyme drug rational

abstract: Bacterial resistance to the aminoglycoside antibiotics is a major health concern because of the elimination of a therapeutic option for the treatment of nosocomial infections. Clinical resistance is commonly caused by the acquisition of genes that encode an aminoglycoside modifying enzyme. These enzymes offer a potential therapeutic target in the fight against aminoglycoside resistance. By gaining a structural understanding of these enzymes the potential is created for rational drug design. The research presented here deals with structural studies on two aminoglycoside resistance enzymes. First the initial stages of structural determination for the bifunctional Aminoglycoside 6'-N-Acetyl transferase Aminoglycoside 2''-O-Phosphotransferase (AAC(6')-APH(2")) including the optimization of the purification procedure for this enzyme. The second enzyme is the Aminoglycoside 3'-O-Phosphotransferase (APH(3')IIIa). Computational studies on this enzyme have been carried out in order to determine models for aminoglycoside binding and also to search for potential enzyme inhibitors. The molecular docking studies for both the aminoglycoside binding and inhibitor search involved the development of a number of novel methods to improve the chance of obtaining a correct model, and to aid in the analysis of the data from the docking studies. These methods have the potential to be applied in future structure based drug design

abstract: Thesis

abstract: Master of Science (MS)


title: Crystal Structure of an Aminoglycoside 6'-N-Acetyltransferase

title: Crystal Structure of AAC(6')-Ii

subject: crystal aminoglycoside acetyltransferase acetyl

abstract: The overwhelming increase in antibiotic resistant bacterial strains poses a serious public health problem, with multiply-resistant strains becoming an important cause of mortality in hospitals. The predominant mechanism of resistance to aminoglycoside antibiotics involves enzymatic modification of the drug, rendering it ineffective. The crystal structure of the aminoglycoside-modifying enzyme aminoglycoside acetyltransferase(6')-Ii (AAC(6')-Ii) in complex with its cofactor, acetyl coenzyme A (AcCoA), was determined at 2.7 Å resolution by the multiwavelength anomalous diffraction technique. The resolution of this structure was subsequently extended to 2.15 Å by molecular replacement, with no significant changes in the topology of the complex. The enzyme was found to exhibit a novel CoA-binding fold, with the cofactor bound in a cleft between the N-and C-terminal arms of the protein molecule. Although the enzyme packs as a monomer in the I4₁32 crystal form, the most probable physiological dimer of the complex was determined through analysis of a number of symmetry-related molecules. The crystal structure of the AAC(6')-Ii•AcCoA complex was compared to the structures of three members of a large superfamily of GCN5-related 𝘕-acetyltransferases (GNATs), namely yeast histone acetyltransferase HAT1, 𝘕-myristoyltransferase, and aminoglycoside acetyltransferase(3)-Ia. Despite negligible sequence similarity between these GNAT superfamily members, a distinct folding pattern is conserved in all four structures. This establishes AAC(6')-Ii as a structural homolog of enzymes with protein acetylating activity, supporting the hypothesis that the enzyme may possess another physiological function in 𝘌𝘯𝘵𝘦𝘳𝘰𝘤𝘰𝘤𝘤𝘶𝘴 𝘧𝘢𝘦𝘤𝘪𝘶𝘮.

abstract: Thesis

abstract: Master of Science (MS)


title: An Interactive System for Tree Alignment and Reconstruction

contributor:
Jiang, Tao
Computation

subject:
tree
alignment
reconstruction
tree alignment
interactive

abstract: This application software system of Tree Alignment and Reconstruction (TAAR) is an X Window based interactive system developed mainly for sequence analysis. It provides a user-friendly graphical interface and convenient operations for performing Pairwise Alignment, 3-Star Alignment, Phylogeny Reconstruction and Generalized Tree Alignment algorithms. The algorithms for Phylogeny Reconstruction and Generalized Tree Alignment are designed based on heuristic stepwise addition and internal node sequence alignment induction methods. All above algorithms are space-efficiently implemented. For each algorithm, both fast and optimal versions are provided for user's convenience. TAAR can be used for DNA, RNA and PROTEIN sequences analysis. In general, as long as the sequence characters are in the range of a-z and A-Z, and the score matrix alphabet contains the sequence alphabet, the sequence would be accepted by this application and all calculations could be carried out. This software system is programmed in C, Motif and X Window Library functions. It is intended to be running in a UNIX and X Window environment.

abstract: Thesis

abstract: Master of Science (MS)


title: Structural Studies of the Bacteriophage Lambda Lysozyme Complexed with a Chitohexasaccharide

title: The Structure of λ Lysozyme-Chitohexasaccharide Complex

contributor:
Berghuis, A. M.
Biochemistry

subject: lysozymne chitohexasaccharide bacteriophage structure

abstract: Lysozyme from the bacteriophage lambda, 1aL, complexed with a chitohexasaccharide has been solved to 2.6 Å by molecular replacement using a mutant form of 1aL as a model. The protein packs as a dimer in the crystal with the backbones of both monomers being nearly identical. The inhibitor molecule resides in the deep cleft in the middle of the bilobal enzyme. Subsites A to Dare occupied by one (GlcNAc)₆ molecule, while the remaining sites interact with two rings from the adjacent (GlcNAc)₆ molecule. The binding mode of 1aL is compared to other lysozymes (HEWL, HuL, GEWL, T4) and Slt70. Interesting differences are noted in the stacking interactions in ring D and the extensive interactions in ring E. It is hypothesized in the thesis that one possible role of the peptide moiety is to interact with Tyr-132, preventing it from forming stacking interactions with ring D, allowing the sugar to penetrate deeper into the active site. Ring E is buried deeply in the enzyme and has a low thermal factor. In addition, the active site is much narrower in 1aL than in other lysozyme structures. A possible explanation has been suggested that rings E and F stay in the active site longer than those in lysozyme to prevent water molecules instead of the O6 atom of ring D to participate in the nucleophilic attack at the end of the reaction.

abstract: Thesis

abstract: Master of Science (MS)


title: Implementation of ^153Sm in Dual Photon Absorptiometry for the Detection of BMC

contributor:
Kennett, T. J.
Engineering Physics

subject: photon abrosptiometry BMC 153Sm

abstract: A ¹⁵³Sm source was used with a dual photon absorptiometry technique in vitro to determine the feasibility of using this isotope as an alternative source to ¹⁵³Gd for further in vivo studies. The source activity was typically .4 Ci (i.e., at the source collimator exit) with a half life of 46.8 hours and photon energies of "103" keV and ¹⁵³Eu X-rays at "42.5" keV. The system was evaluted using phantom measurements of water, aluminum and polyethylene to simulate soft tissue, bone and fat respectively. During stationary measurements, a total absorber thickness between 15 and 22.5 cm. was used in conjunction with an aluminum thickness range of .314 to 1.91 em. (.848-5.16 g/cm²). The coefficient of variation and the accuracy at most total absorber thicknesses was less than 2%. This showed that ¹⁵³Sm could produce precision and accuracy comparable to ¹⁵³Gd. When polyethylene (fat) was present, as the amount of polyethylene in the RST measurement increased, the error due to a difference in the amount of polyethylene between the RST and the BMC measurements increased. For a percent difference in polyethylene thickness between the RST and BMC measurement of less than 50% at 16.3% RST polyethylene content, measurements of aluminum (bone) above 1.8 g/cm²(.662 cm. of aluminum) produced results accurate to within 10% of the true aluminum (BMC) value for this experimental geometry. Motional studies were performed using a modified OHIO NUCLEAR rectilinear scanner. The optimal scan speed was shown to be in the range of 4-6 mm./sec. with a sample space of between .5 to .7 cm.. The worst-case dose using the optimal scan parameters was determined as 6.2 and 1. 4 mRad per scan of approximately 24 minutes for bone and soft tissue respectively. The combination of stationary and motional in vitro studies indicated that ¹⁵³Sm could be an economical and effective alternative to ¹⁵³Gd for clinical studies. A Monte Carlo simulation was implemented to determine the optimal detector collimator entrance size. Simulation of the experimental geometry indicated that the .6 cm. detector collimation used experimentally was near the optimal size to produce efficient results in terms of accuracy and precision. A brief summary of Monte Carlo methods and radiation theory is also included.

abstract: Thesis

abstract: Master of Engineering (ME)


title: Application of First Order Unimolecular Rate Kinetics to Interstitial Laser Photocoagulation

contributor:
Wyman, Douglas
Medical Physics

subject:
first order
unimolecule
kinetics
laser
photocoagulation

abstract: An investigation of the temperature response and corresponding lesion growth resulting from in vivo interstitial laser photocoagulation was performed in order to test the applicability of Arrhenius theory. The irradiations were performed in vivo in rabbit muscle for various exposures at 1.0W using an 805 nm diode laser source coupled to an optical fibre with a pre-charred tip, thereby forcing it to function as a point heat source. Temperature responses were measured using a five-microthermocouple array along a range of radial distances from the point heat source. Each temperature profile was fitted with a curve predicted by the Weinbaum-Jiji bioheat transfer equation. The lesions were resected 48 hours after irradiation and the boundary of thermal damage resulting in necrosis was determined histologically. Numerical integration of the Arrhenius integral using temperature-time data at the lesion boundary produced corresponding activation energy and pre-exponential factor pairs (Eₐ , α) consistent with reported values for various other endpoints and tissue types. As well, theoretical predictions of the lesion growth from Arrhenius theory agreed well with experimental results. However, the thermal parameters, which are generally assumed to be constant when solving the bioheat transfer equation, were found to vary with radial distance from the source, presumably due to dependence on temperature.

abstract: Thesis

abstract: Master of Science (MS)


title: GUT SEROTONIN: REVEALING ITS ROLE IN ANTIMICROBIAL PEPTIDE PRODUCTION

contributor:
Khan, Waliul I
Medical Sciences

subject: Serotonin

subject: Inflammatory Bowel Disease (IBD)

subject: β-defensin

abstract: Serotonin (5-hydroxytryptamine [5-HT]) is a key enteric signaling molecule that is implicated in many gastrointestinal (GI) disorders, including inflammatory bowel disease (IBD). Enterochromaffin (EC) cells are a key subgroup of enteric endocrine cells and produce the majority of 5-HT via tryptophan hydroxylase 1 (Tph1) in the gut. Recently, we have identified a pivotal role of 5-HT in the pathogenesis of experimental colitis, whereby 5-HT plays as a pro-inflammatory molecule. Gut function as well as pathology rely on interactions with gut microbiota. The intestinal epithelial cells produce antimicrobial peptides (AMPs), maintaining the mucosal barrier by shaping gut microbiota composition. Among the AMPs, β-defensins are the most well investigated subtype in the colon. Aberrant β-defensin expression has been reported in association with various GI disease pathogenesis including IBD. As EC cells are dispersed throughout the intestinal epithelium, it seems possible that 5-HT can modify β-defensin production which can regulate gut inflammation by influencing gut microbial composition. Colitis was induced with dextran sulfate sodium (DSS) in Tph1+/+ and Tph1-/- (which have lower amounts of 5-HT in gut). Tph1-/- mice exhibited higher levels of β-defensin in the colon, compared with wild-type littermates post-DSS. In addition, increased expression of β-defensin in Tph1-/- mice was suppressed by 5-hydroxytryptophan (5-HTP; precursor of 5-HT) treatment. 5-HT treatment resulted in decreased human β-defensin (hBD) 1 and hBD-2 expression in HT-29 cells. Peroxisome proliferator-activated receptor gamma (PPAR-γ) is essential for maintaining β-defensin expression in the colon. GW-9662, PPAR-γ antagonist, reduced mouse β-defensin (mBD) 1 and mBD-3 (orthologue of hBD-2). Furthermore, disrupting 5-HT7 receptors, but not 5-HT3 or 5-HT4, led to enhanced expression of PPAR-γ via ERK1/2-dependent mechanism. These observations provide us with novel information on pivotal role of gut-derived 5-HT in innate immune response and highlight the potential benefits of targeting 5-HT signaling in various GI disorders such as IBD.

abstract: Thesis

abstract: Master of Science (MSc)


title: Statistical Simulation of Patient Compliance

subject: patient compliance simulation

abstract:
Patient compliance is one of the important factors which effect patient outcome in therapeutic trials. As the methods of measuring patient compliance have been developed, the impact of compliance on therapeutic regimen performance could be studied. In this project, patient compliance measures are defined and compliance distributions are introduced. The two are combined from the two sources to improve both the knowledge of drug-outcome combinations and compliance information has been simulated in the case of therapeutic trials. The study of compliance effects on patient outcome was approached in two ways; looking at the fixed compliance effects upon the entire dose-response curve, and looking at the compliance measure as a variable effect on dose-response outcome of patients, the latter by means of statistical simulation. It was shown that patient compliance information affected patient outcome in terms of the achievement of the therapeutic goal. Also, the results of this project support what should be said about the
basis of drug-outcome combinations and prescriptions, when we mention patient compliance. Another result is that Compliance-Response-outcome chains were obtained in terms of percentages to respond from simulation of some common design models used in therapeutic trials.

abstract: Thesis

abstract: Master of Science (MS)


title: Stationary Phase Expression of the Arginine Biosynthetic Operon (ARGCBH) in Escherichia Coli

title: Stationary Phase Expression of ARGCBH in Escherichia Coli

contributor:
Schellhorn, Herb
Biology

subject:
e coli
arginine
biosynthetic
operon

abstract: In this study, we report that expression of the 𝘢𝘳𝘨𝘊𝘉𝘏 operon is induced in stationary phase cultures and that this increase is largely dependent on RpoS, the alternative stress sigma factor. Using combinatorial 𝘢𝘳𝘨𝘙 and 𝘳𝘱𝘰𝘚 mutants, we evaluated the relative contributions of these two regulators to the expression of 𝘢𝘳𝘨𝘏 using operon 𝘭𝘢𝘤𝘡 fusions. While ArgR was found to be the main factor responsible for de-repression of the 𝘢𝘳𝘨𝘊𝘉𝘏 operon, RpoS was required for full expression of this biosynthetic operon at concentrations below 10 μg arginine ml⁻¹, a level at which growth of an arginine auxotroph was arginine limited. At high arginine concentrations (>10 μg ml⁻¹) 𝘢𝘳𝘨𝘊𝘉𝘏 expression was strongly repressed as expected by ArgR. 𝘢𝘳𝘨𝘊𝘉𝘏 expression was 30 fold higher in Δ𝘢𝘳𝘨𝘙 mutants relative to a wild type fully repressed strain and this expression was independent of RpoS. These results indicate that RpoS plays an important role in the regulation of arginine biosynthesis, particularly when the operon is partially de-repressed as would be the case in starvation conditions.

abstract: Thesis

abstract: Master of Science (MS)


title: A Client-Centered Care Curriculum for Case Managers

contributor:
Rideout, Elizabeth
Richardson, Jack

subject: client curriculum case manager

abstract: The purpose of this project is to improve case managers' personal effectiveness with clients, and to foster client-centred care. The project describes the plan for the development, implementation, and evaluation of a client-centred care curriculum for community case managers at the Hamilton-Wentworth Community Care Access Centre (CCAC). Client-centred care is really about how we treat one another, and is supported in the literature. For this project, client-centred care means: honoring personhood, communicating therapeutically, being reflective, setting goals with clients, and therapeutic use of self. The concept of therapeutic use of self is about how one uses oneself to help clients (Whall, 1988). The project describes implementation and evaluation of a therapeutic use of self workshop which is one component of the entire curriculum. The curriculum is guided by principles of adult learning, including self-directed learning, and transformative learning. The guiding theoretical frameworks for this project include: Miller and Seller's (1990) transaction and transformation education positions, the Leithwood-lnnovations Profile For Implementation (as cited in Miller & Seller, 1990), and Patton's (1997) Utilization-Focused Evaluation. Although this project has the potential to make a significant impact on case managers and clients, the curriculum has yet to be tested. Recommendations for use of the curriculum within the CCAC are provided.

abstract: Thesis

abstract: Master of Science (Teaching)


title: "Platelet Adhesion and Activation on Fluoropolymers"

contributor:
Feuerstein, I. A.
Chemical Engineering

subject: platelet fluoropolymers

abstract: Investigation of platelet adhesion, platelet activation, and platelet morphology was performed on Hexafluoroethane coated glass coverslips, Fluorinated Ethylene Propylene (FEP), and Polymethyl Methacrylate (PMMA) thin film polymers. Hexafluoroethane was coated on glass coverslips using Radio Frequency Glow Discharge (RFGD) polymer deposition and was supplied by researchers at the University of Washington in Seattle. For several years now, analogously prepared Fluoropolymer surfaces have been reported to exhibit unique protein adsorption properties and reduced adherent platelet concentrations compared to standard fluoropolymers and non-fluorinated polymers (Kiaei, 1995). Platelet interactions were assessed using a novel, dynamic, and physiologically relevant experimental system. In-vitro tests were performed on polymer surfaces mounted in a light transparent flow cell by pumping whole blood through the apparatus to deposit platelets over the polymer surface. Red blood cells were rinsed from the flow cell with plasma immediately after platelet deposition to permit observation of adherent cells. Dynamic, real time single cell morphology observation was made under low flowing plasma conditions using light microscopy and recorded using a computer image acquisition system. Physiologic conditions were maintained using a low plasma flow rate which ensured available nutrients for platelets as tests were performed for up to 90 minutes. Compared to PMMA and FEP, Hexafluoroethane surfaces exhibited the lowest platelet surface concentrations with overall mean adherent platelet concentrations of 8165, 6895, and 4387 platelets per mm², respectively. PMMA is a more activating surface compared to the fluoropolymers tested, however, the rate of progress of platelet activation and morphological trends are similar between the Hexafluoroethane and FEP polymer surfaces. Activation parameters, a quantification of the state of platelet activation, support experimental observations made concerning morphological change information on Hexafluoroetbane, FEP, and PMMA. A Scanning Electron Microscopy study involving all three test surfaces, fixed after 60 or 90 minute plasma flow maintained experiments, support the hypothesis that the pancake platelet evolves from a spreading platelet. Routinely observed, pancake platelets are circular, 3-5 μm in diameter, and have round raised protuberances at the periphery of the cell membrane. Images showing several stages of spread cell retraction on Fluoropolymers tested without added thrombin stimulant give greater detail of peripheral fragmentation and further support a mechanism for polymer surface induced platelet microparticle formation.

abstract: Thesis

abstract: Master of Engineering (ME)


title: Assisted Suicide and the Suicide Stigma

contributor:
Boetzkes, E.
Philosophy

subject:
assisted suicide
suicide
stigma

abstract: The purpose of this thesis is to argue for the permissibility of assisted suicide in certain cases. Since the practice of assisted suicide involves the act of suicide, I have chosen to focus my arguments on a defense of the act of suicide in certain cases. I then argue that there is no moral difference between suicide and assisted suicide, so that in most cases if it is permissible for a person to take his or her own life then it ought to be permissible for him or her to receive suicide assistance. I accomplish this first by critically analzying the psychological view of suicide, which gives to rise to the incompetency argument, and by refuting numerous other arguments offered to demonstrate the unconditional moral impermissibility of suicide. I then defend suicide as being morally permissible if 1) the agent is competent; and 2) the suicide does not violate any overriding obligations that would not otherwise be violated. I also defend a notion of 'full permissibility', meaning an action that a person ought to be free to perform without justified paternalistic interference from others. An action, and thus a suicide, is fully permissible if it is morally permissible as well as rational for the person in question. In the final chapter I make the move from defending suicide in certain cases if it is permissible for a person to take his or her own life then it also ought to be permissible for him or her to receive assistance.

abstract: Thesis

abstract: Master of Arts (MA)


title: A Multiple-Source Delivery System for Interstitial Laser Photocoagulation

contributor:
Wyman, Douglas
Physics

subject: coagulation interstitial laser delivery

abstract: Interstitial laser photocoagulation (ILP) is a minimally invasive technique for destroying solid, localised tumours thermally by delivering infrared laser energy directly into the targeted volume via percutaneously implanted optical fibres. Using current treatment parameters, each fibre delivers sufficient energy to destroy a volume of one to two cm³; larger lesions may be created by using multiple fibres excited simultaneously. An efficient delivery system has been constructed consisting of several fibres bundled through a single cannula and splayed out through a specially designed tip. This delivery system is simple to use and provides accurate fire placement. By linearly superposing single source solutions to the bioheat transfer equation, a mathematical model for coalescent thermal lesions has been developed. It has been determined that large, clinically useful thermal lesions can be created by implanting four sources at the corners of a square. It has been demonstrated, through 𝘦𝘹 𝘷𝘪𝘷𝘰 experimentation in bovine liver, that the model correctly predicts the dimensions of the thermal lesions.

abstract: Thesis

abstract: Master of Science (MS)


title: Surface and Biological Effects of Peptide Orientation Evaluated Using Gold-Coated Polyurethanes

contributor:
Sheardown, Heather
Chemical Engineering

subject: peptide gold polyurethanes

abstract: Cell adhesion and growth are central issues in the otherwise promising method of endothelialization of materials for improving blood compatibility. To improve cell adhesion to biomaterial surfaces, surface modification with cell adhesion peptides is often used. In particular, arginine-glycine-aspartic acid (RGD), long recognized as a peptide sequence that plays and important role in cell adhesion, has been covalently attached to surfaces to enhance cell adhesion. In this work, the effect of the orientation of the cell adhesion peptide immobilized on the surface was studied through using gold surfaces, which can be readily modified with thiols and sulfur-containing groups. Peptide orientation was controlled by the placement of the cysteine (C) at either the C- or N-terminus, Two cell adhesion peptides, the non-specific RGD and more endothelial cell specific REDV (arginine-glutamic acid-aspartic acid-valine), were studied for their effect on the surface chemical and biological properties, including effects on the interactions with the endothelial cell line ECV304. Vitronectin adsorption to the modified surfaces was specifically examined as a possible reason for differences noted. The results suggest that peptide orientation plays an important role in the interactions of cells and proteins to the modified surfaces. Peptides with the cysteine at the N-terminus showed increased adhesion of endothelial cells from the ECV304 line, with the greatest adhesion noted consistently on the CREDV-modified surfaces. Differences in surface chemistry as evaluated by x-ray photoelectron spectroscopy were also found higher levels of bonded peptide when the thiol-containing cysteine was in the N-terminal position. These results suggest that the secondary structure of the peptide can be used to enhance or to limit its reaction with the surface. Furthermore, while cell adhesion was noted during culture in the absence of serum, significant increases in the numbers of adherent cells were noted on all surfaces when the cells were grown in the presence of serum. Immunoblotting and culture with antibodies demonstrated that this increase in the adhesion of the cells is likely mediated primarily by the cell adhesion peptide vitronectin.

abstract: Thesis

abstract: Master of Engineering (ME)