Effect of citrate on the redox properties of cytochrome C and its kinetic and binding behaviour with cytochrome oxidase /

Increasing citrate concentration, at constant ionic strength (30 mM) decreases the rate of cytochrome ~ reduction by ascorbate. This effect is also seen at both high (600 mM) and low (19 mM) ionic strengths, and the Kapp for citrate increases with increasing ionic strength. Citrate binds d both ferri -and ferrocytochrome ~, but with a lower affinity for the latter form (Kox . .red d = 2 mM, Kd = 8 mM) as shown by an equilibrium assay with N,N,N',N', Tetramethyl E- phenylenediamine. The reaction of ferricytochrome ~with cyanide is also altered in the presence of citrate: citrate increases the K~PP for cyanide. Column chromatography of cytochrome ~-cytochrome oxidase mixtures shows citrate increases the dissociation constant of the complex. These results are confirmed in kinetic assays for the "loose"site (Km = 20 pM) only. The effect of increasing citrate observable at the "tight" site (Km = 0.25 pM) is on the turnover number and not on the K . These results suggest a mechanism m where anion binding to cytochrome £ at the tight site affects the equilibrium between two forms of cytochrome c bound cytochrome oxidase: an active and an inactive one.

Synthesis of fluorescent analogues of a-tocopherol as ligands for the human a-tocopherol transfer protein (a-TTP) /

To further understand in vivo localization and trafficking of a-tocopherol (a-Toe), the most biologically active form of vitamin E, between lipid environments, tocopherols are required that can be followed by teclu1iques such as confocal microscopy and fluorescence resonance energy transfer (FRET) assays. To this end, sixteen fluorescent analogues of a-tocopherol (la-d [(1)anthroy loxy -a-tocopherols, A O-a-Toes], 2a-d [w-nitro benzoxadiazole-a-tocopherols, NBD-aToes], 3a-d [w-dansyl-a-tocopherols, DAN-a-Toes], and 4a-d [w-N-methylanthranilamide-atocopherols, NMA-a-TocsD were prepared by substituting fluorescent labels at the terminus of w-functionalized alkyl chains extending from C-2 of the chroman ring while retaining key binding features of the natural ligand. These compounds were prepared starting from (S)-Trolox® acid VIa esterification, protection, and reduction producing the silyl-protected (S)-Trolox aldehyde that was coupled using Wittig chemistry to different w-hydroxyalkylphosphonium bromides. Reduction of the alkene generated the w-hydroxy functionalized 2-n-alkyl intermediates 9a-d having the necessary 2R stereochemistry. A series of functional group manipulations including mesylation, substitution with azide, and hydride reduction provided w-amino functionalized intermediates 12a-d as well. Coupling intermediates 9a-d and 12a-d with the selected fluorophores (9- anthracene carboxylic acid, 4-chloro-7-nitrobenz-2-oxa-l,3-diazole, 5- dimethylaminonapthalene-l-sulfonyl chloride, and I-methyl-2H-3,1-benzoxazine-2,4(1H)dione), followed by deprotection of the phenolic silyl group, gave the desired fluorescent ligands la-d, 2a-d, 3a-d and 4a-d in good yield. Assessment of their binding affinities with recombinant human a-tocopherol transfer protein (ha-TTP) utilizing fluorescent titration binding assays identified competent ligands for further use in protein studies. Compounds Id (C9-AO-a-Toc) and 2d (C9-NBD-a-Toc) both having nonyl alkyl chain extensions between the chromanol and fluorophore were shown to bind specifically to ha-TTP with dissociation constants (KdS) of approximately 280 nM and 55 nM respectively, as compared to 25 nM for the natural ligand 2R,4'R,^'R-a-tocophQxoL.

Primary characterization of genes involved in the colony development of the insect pathogenic fungus Metarhizium anisopliae /

Strain improvement of the insect pathogenic fungus Metarhizium anisopUae is necessary to increase its virulence towards agricultural pests and thus improve its commercial efficacy. Nevertheless, the release of genetically modified conidia in crop fields may negatively affect the ecosystem. Controlling conidiation is a potential means of limiting the release of engineered strains since conidia are the infective propagules and the means of dispersal. The purpose of this study was to research the colony development of M. anisopUae to identify potential targets for genetic manipulation to control conidiation. Following Agrobacterium tumefaciem insertional mutagenesis, phenotypic mutants were characterized using Y-shaped adaptor dependent extension PCR. Four of 1 8 colony development recombinants had T-DNA flanking sequences with high homology to genes encoding known signaling pathway proteins that regulate pathogenesis and/or asexual development in filamentous fungi. Conidial density counts and insect bioassays suggested that a Serine/Threonine protein kinase COTl homolog is not essential for conidiation or virulence. Furthermore, a choline kinase homolog is important for conidiation, but not virulence. Finally, the regulator of G protein signaling CAG8 and a NADPH oxidase NoxA homolog are necessary for conidiation and virulence. These genes are candidates for further investigation into the regulatory pathways controlling conidiation to yield insight into promising gene targets for biocontrol strain improvement.

Water quality and fish populations of the Welland River, Ontario /

The water quality and fish populations of the Welland River were observed to decline with distance downstream. This coincided with increased agricultural , domestic and industrial waste loadings. The river upstream of the City of Welland received considerable loadings from agricultural sources. Centrarchids, sciaenids, ictalurids, cyprinids and esocids characterized this upper section of the river. Most of these species were tolerant of low dissolved oxygen concentrations and the high turbidity which prevailed there . The river near Port Robinson receives many industrial and domestic wastes as evidenced by the water quality data. The fish in this section were less abundant and the observed population was comprised almost solely of cyprinids. Further downstream, near Montrose, the Welland River received shock loads of chemical wastes that exceeded a specific conductance of ISiOOO ;umhos/cm. Few fish were captured at this site and those that were captured were considered to be transients. A review of the literature revealed that none of the common indices of water quality in use today could adequately predict the observed distributions. In addition to the above, the long-term trend (l3 yrs) of water quality of the lower Welland River revealed a gradual improvement. The major factor thought to be responsible for this improvement was the operation of the Welland Sewage Treatment Plant. The construction of the New Welland Ship Canal coincided with large fluctuations of the total solids and other parameters downstream. These conditions prevailed for a maximum of three years (1972- 1975)' Furthermore, spawning times and temperatures, geographic distributions, length-weight regressions and many other descriptive aspects of the ecology of some 26 species/ taxa of fish were obtained. Several of these species are rare or new to southern Ontario.

Synthesis of chiral homoallylic alcohols and phthalans through the asymmetric allylation of carbonyl compounds /

The implementation of chiral centres within biologically active compounds has been a perplexing yet motivational force in chemistry. This work presents the attempted formation of a concurrent or sequential tandem catalyzed methodology of enantioselective nucleophilic addition and electrophilic cyclization. The 2'- arylalkynyl- aldehyde, ketone, and imine substrates used within were adeptly chosen with a dually activated structure; 1) for nucleophilic addition to the electrophilic substituents; and 2) for carbophilic activation of the alkyne substituent to undergo cyclization. To accomplish the nucleophilic addition, two distinct allylation methodologies were pursued: (/?)-BINOL catalyzed-allylboration and (5)- BINAP-AgF catalyzed-allylsilylation. BINAP catalyzed enantioselective allylation of 2'-arylalkynyl-aldehydes, to form chiral homoallylic alcohols, was successful. Homoallylic alcohols were isolated with high enantio-purity (>80%), which then underwent sequential cyclization to form chiral allylic phthalans, in moderate yields. An application of this methodology towards the construction of biologically active compounds was included with the partial synthesis of the natural product and H. pylori inhibitor, (+)-Spirolaxine methyl ether.

Significance of DNA base excision repair in the maintenance of mitochondrial function in Saccharomyces cerevisiae /

Mitochondria have an important role in cell metabolism, being the major site of ATP production via oxidative phosphorylation (OXPHOS). Accumulation of mtDNA mutations have been linked to the development of respiratory dysfunction, apoptosis, and aging. Base excision repair (BER) is the major and the only certain repair pathway existing in mitochondria that is in responsible for removing and repairing various base modifications as well as abasic sites (AP sites). In this research, Saccharomyces cerevisiae (S. cerevisiae) BER gene knockout strains, including 3 single DNA glycosylase gene knockout strains and Ap endonuclease (Apn 1 p) knockout strain were used to examine the importance of this DNA repair pathway to the maintenance of respiratory function. Here, I show that individual DNA glycosylases are nonessential in maintenance of normal function in yeast mitochondria, corroborating with previous research in mammalian experimental models. The yeast strain lacking Apn 1 p activity exhibits respiratory deficits, including inefficient and significantly low intracellular ATP level, which maybe due to partial uncoupling of OXPHOS. Growth of this yeast strain on respiratory medium is inhibited, but no evidence was found for increased ROS level in Apn 1 p mitochondria. This strain also shows an increased cell size, and this observation combined with an uncoupled OXPHOS may indicate a premature aging in the Apnlp knockout strain, but more evidence is needed to support this hypothesis. However, the BER is necessary for maintenance of mitochondrial function in respiring S.cerevisiae.

Some ecological effects of beaver upon the watersheds in the Porcupine Hills, Alberta /

This ecological investigation of the beaver (Castor canadensis canadensis Kuhl) was part of the Eastern Slopes (Alberta) Watershed Research program conducted during the summers of 1968 . 1969 . 1970. and 1971 . A soil particle size analysis indicated that soil heavy with clay was used by the beaver for construction purposes in the Porcupine Hills . Examples were given of the beaver controlling erosion with the construction of dams . and also causing erosion to occur . However . in general . the beaver slow down soil erosion and decrease the loss of soil from this region. The beaver utilized measureable amounts of herbaceous vegetation . However, the utilization of herbs by the beaver requires further investigation. A system of ex~sures and enclosures of herbaceous vegetation plots was used to study the utilization of sedges , grasses , and forbs . The beaver indicated stronger species preference for willow as compared to aspen . The size preference for willow was in the 1.0 to 10.0 centimeters basal diameter classes , while the beaver utilization of aspen indicated a preference for the largest trees with basal diameter greater than 20.0 centimeters. Willow was the most important plant in the low lying areas with regard to distribution, abundance and to produce sustained yield. The beaver used this plant for food and construction throughout the study area. The distribution of aspen was limited. and this species did not appear to produce more than one crop in the lifetime of a beaver colony. Nine out of 15 woody plant types were sampled by the beaver in this region. A plot-intercept transect technique was used for systematic vegetation sampling of the woody vegetation in six intensively studied watersheds. The beaver population of the Porcupine Hills region of SW-Alberta is believed to depend upon the chinooks. During the 1971 ground census, a total of 60 active beaver colonies were tallied on the 930 square kilometers large study area. The beaver of the region were not found to store large food caches during fall and winter, however, they are believed to collect feed periodically throughout the winter months. It was observed that the severe winter in 1968-69, reduced by 27 per cent the number of active beaver colonies within the study area. The Porcupine Hills region had 0.07 beaver colony per square kilometer in 1971, a low density of beaver colonies due to the rough topography of the area. However, the importance of the beaver ponds was somewhat clarified as they provide increased moisture, which lessens the fire damage, and store water for wildlife and cattle in the area. Meteorological data was collected by the author in collaboration with the Department of Transport.

A preliminary study of the MDH variability in Lemna minor - Lemna turionifera /

Lemna minor is a small aquatic polyploid angiosperm which reproduces apomictically and has a worldwide distribution. This study was vmdertaken to characterize the extent and nature of phenotypic variability. The techniques of starch gel electrophoresis were used in this investigation and. MDH phenotypes of several populations from Ontario, USA and Africa were examined and compared. Heat stability, molecular weight and cell fractionation analyses were also done to identify locus specific MDH bands. The results of the population surveys suggest that there is little genetic variability present both within and between Lemna minor/Lemna turionifera . Evidence of correlation of physiological and seasonal variation patterns was found.

Changes in the magnitude and pattern of translocation of photoassimilated ¹CO in soybean plants following an acute exposure to gamma radiation /

Young soybean plants (Glycine ~. L. cultivar Harosoy '63), grown under controlled conditions, were exposed to gamma radiation on a single occasion. One hour following exposure to 3,750 rads, the mature trifoliate leaf of the soybean plant was isolated in a closed system and permitted to photoassimilate approximately 1-5 pCi of 14C02 for 15 minutes. After an additional 45 minute-period, the plant was sacrificed and the magnitude of translocation and distribution pattern of 14C determined. In the non-irradiated plants 18~ of the total 14C recovered was outside the fed leaf blades and of this translocated 14c, 28~ was above the node of the fed leaf, 38~ in the stem below the node, 28~ in the roots and 7~ in the petiole. As well, in the irradiated plants, a smaller per cent (6~) of the total 14 C recovered was exported out of the source leaf blades. Of this translocated 14c , a smaller per cent (20~) was found in the apical region above the node of the source leaf and a higher per cent (45~) was recovered from the stem below the node and in the petiole (11~). The per cent of exported 14 C recovered from the root was unaffected by the radiation. Replacement of the shoot apex with 20 ppm IAA immediately following irradiation, only J partially increased the magnitude of translocation but did completely restore the pattern of distribution to that observed in the non-irradiated plants. From supplementary studies showing a radiationinduced reduction of photosynthetic rates in the source leaf and a reduction of the cumulative stem and leaf lengths in the apical sink region, the observed effects of radiation on the translocation process have been correlated to damage incurred by the source and sink regions. These data suggest that the reduction in the magnitude of translocation is the result of damage to both the source and sink regions rather than the phloem conducting tissue itself, whereas the change in the pattern of translocation is probably the result of a reduced rate of 14C-assimilate movement caused by a radiation-induced decrease of sink metabolism, especially the decrease in the metabolism of the apical sink.

The structural effects of divalent cations and insulin on phospholipid model membranes /

It is well accepted that structural studies with model membranes are of considerable value in understanding the structure of biological membranes. Many studies with models of pure phospholipids have been done; but the effects of divalent cations and protein on these models would make these studies more applicable to intact membrane. The present study, performed with above view, is a structural analysis of divalent io~cardio1ipin complexes using the technique of x-ray diffraction. Cardiolipin, precipitated from dilute solution by divalent ionscalcium, magnesium and barium, contains little water and the structure formed is similar to the structure of pure cardiolipin with low water content. The calcium-cardiolipin complex forms a pure hexagonal type II phase that exists from 40 to 400 C. The molar ratio of calcium and cardiolipin in the complex is 1 : 1. Cardiolipin, precipitated with magnesium and barium forms two co-existing phases, lamellar and hexagonal, the relative quantity of the two phases being dependent on temperature. The hexagonal phase type II consisting of water filled channels formed by adding calcium to cardiolipin may have a remarkable permeability property in intact membrane. Pure cardiolipin and insulin at pH 3.0 and 4.0 precipitate but form no organised structure. Lecithin/cardiolipin and insulin precipitated at pH 3.0 give a pure lamellar phase. As the lecithin/cardiolipin molar ratio changes from 93/7 to SO/50, (a) the repeat distance of the lamellar changes from 72.8 X to 68.2 A; (b) the amount of protein bound increases in such a way that cardiolipin/insulin molar ratio in the complex reaches a maximum constant value at lecithin/cardiolipin molar ratio 70/30. A structural model based on these data shows that the molecular arrangement of lipid and protein is a lipid bilayer coated with protein molecules. The lipid-protein interaction is chiefly electrostatic and little, if any, hydrophobic bonding occurs in this particular system. So, the proposed model is essentially the same as Davson-Daniellifs model of biological membrane.

Paleolimnology of selected Precambrian Shield lakes /

The analysis of fossil diatoms and Mallomonas assemblages in a 2.85 m sediment core revealed that a series of distinct floristic changes have occurred in the development of Found Lake, a small Shield lake in southern Ontario. Climatic and vegetational changes in the lake's watershed were closely associated with successional changes in the lake's biota. Nutrients released by the deciduous component of the Found Lake watershed appeared to be especially important in determining diatom and Mallomonas standing crop. The top 20 cm of sediment of 3 ,Shield lakes was then investigated using close interval (1 cm) analyses of diatoms, Mallomonas scales, pollen grains and sedimentary phosphorus. Found and Jake Lake are adjacent to Highway 60, whereas Delano Lake has been undisturbed and was used as a control. Dramatic changes in the diatom and Mallomonas communities were recorded in the Found and Jake Lake stratigraphies and could be closely associated with known historical events. Increased turbidity and nutrient enrichment were believed responsible for these successional changes. In addition, diatom and Mallomonas standing crop increased substantially following road construction in Found Lake's drainage basin. Meanwhile, no. sharp changes in diatom or Mallomonas communities were recorded in the recent sediments of the control (Delano) lake. The use£ulness o£ Synuracean scales as paleoindicators, as well as the importance o£ sectioning cores at close intervals during transition periods in a laker's development, was stressed.

Perturbing the impulse activity of a single identified neuron augments the formation of long-term memory in a molluscan semi-intact preparation /

The aim of this study was to investigate the neural correlates of operant conditioning in a semi-intact preparation of the pond snail, Lymnaea stagnalis. Lymnaea learns, via operant conditioning, to reduce its aerial respiratory behaviour in response to an aversive tactile stimulus to its open pneumostome. This thesis demonstrates the successful conditioning of na'ive semiintact preparations to show learning in the dish. Furthermore, these conditioned preparations show long-term memory that persists for at least 18 hours. As the neurons that generate this behaviour have been previously identified I can, for the first time, monitor neural activity during both learning and long-term memory consolidation in the same preparation. In particular, I record from the respiratory neuron Right Pedal Dorsal 1 (RPeD 1) which is part of the respiratory central pattern generator. In this study, I demonstrate that preventing RPeDl impulse activity between training sessions reduces the number of sessions needed to produce long-term memory in the present semi-intact preparation.

The effects of yeast inoculation rate and acclimatization to juice on icewine fermentation /

Icewine is an intensely sweet, unique dessert wine fennented from the juice of grapes that have frozen naturally on the vine. The juice pressed from the frozen grapes is highly concentrated, ranging from a minimum of 35° Brix to approximately 42° Brix. Often Icewine fennentations are sluggish, taking months to reach the desired ethanol level, and sometimes become stuck. In 6 addition, Icewines have high levels of volatile acidity. At present, there is no routine method of yeast inoculation for fennenting Icewine. This project investigated two yeast inoculum levels, 0.2 gIL and 0.5 gIL. The fennentation kinetics of inoculating these yeast levels directly into the sterile Icewine juice or conditioning the cells to the high sugar levels using a step wise acclimatization procedure were also compared. The effect of adding GO-FERM, a yeast nutrient, was also assessed. In the sterile fennentations, yeast inoculated at 0.2 gIL stopped fennenting before the required ethanol level was achieved, producing only 7.8% (v/v) and 8.1 % (v/v) ethanol for the direct and conditioned inoculations, respectively. At 0.5 gIL, the stepwise conditioned cells fennented the most sugar, producing 12.2% (v/v) ethanol, whereas the direct inoculum produced 10.5% (v/v) ethanol. The addition of the yeast nutrient GO-FERM increased the rate of biomass accumulation, but reduced the ethanol concentration in wines fennented at 0.5 gIL. There was no significant difference in acetic acid concentration in the final wines across all treatments. Fennentations using unfiltered Icewine juice at the 0.5 gIL inoculum level were also compared to see if the effects of yeast acclimatization and micronutrient addition had the same impact on fennentation kinetics and yeast metabolite production as observed in the sterile-filtered juice fennentations. In addition, a full descriptive analysis of the finished wines was carried out to further assess the impact of yeast inoculation method on Icewine sensory quality. At 0.5 gIL, the stepwise conditioned cells fennented the most sugar, producing 11.5% (v/v) ethanol, whereas the direct inoculum produced 10.0% (v/v) ethanol. The addition of the yeast nutrient GO-FERM increased the peak viable cell numbers, but reduced the ethanol concentration in wines fennented at 0.5 gIL. There was a significant difference 7 in acetic acid concentration in the final wines across all treatments and all treatments affected the sensory profiles of the final wines. Wines produced by direct inoculation were described by grape and raisin aromas and butter flavour. The addition of GO-FERM to the direct inoculation treatment shifted the aroma/flavour profiles to more orange flavour and aroma, and a sweet taste profile. StepWise acclimatizing the cells resulted in wines described more by peach and terpene aroma. The addition of GO-FERM shifted the profile to pineapple and alcohol aromas as well as alcohol flavour. Overall, these results indicate that the addition of GO-FERM and yeast acclimatization shortened the length of fermentation and impacted the sensory profiles of the resultant wines.

Epidemiology and phylogenetic analysis of West Nile virus in Ontario, Canada, 2002-2003 /

Since the discovery of West Nile (WN) virus in the Western Hemisphere many surveillance programs have been implemented to monitor the epidemiology and genetic variation of WN virus in North America. This project was based on the WN virus Adult Mosquito Identification and Diagnostic Program conducted at Brock University for Ontario, Canada, during the 2002 and 2003 transmission seasons. There are three sections to this thesis. The first section investigated which mosquito species carry WN virus in Ontario, Canada throughout the 2002-2003 transmission seasons. It was found that from the 2002 data, eight mosquito species were detected with WN virus (Aedes vexans, Anopheles punctipennis, Coquilleltidia perlurbans, Culex salinarius, Cx. pipiens, Cx. resluans, Ochlerolalus Irivillalus and Och. Iriserialus) and 7.19% of the total mosquito pools tested were found to be WN virus positive (129 positive poolsll, 793 total pools tested). In 2003, WN virus was detected in only five mosquito species (Ae. vexans, Cx. salinarius, Och. Iriserialus, Cx. pipiens and Cx. resluans) and 1.42% of the total mosquito pools tested were WN virus positive (101 positive poolsl7,1 01 total pools tested). WN virus positive mosquito pools were detected 3-4 weeks earlier in 2002 compared to 2003 data. The second section investigated the actual infection rate (IR) of clearly identified Cx. pipiens and Cx. resluans from the 2002 outbreak. It was found that significantly more ex. resluans were infected with WN virus compared to ex. pipiens. The third section investigated the degree of variability of the WN virus genome. A 879 nucleotide section of the WN virus genome was amplified from 21 American Crows and 20 adult female mosquitoes from Ontario, Canada, and compared to the homologous region of the original New York 1999 Chilean Flamingo sequence (NY99FL). Seventy-two nucleotides from Ontario WN virus sequences showed variability compared to NY99FL with 10 synapotypic changes. Phylogenetic analysis revealed a close relationship between Ontario and US WN virus sequences.

The impact of the Multicolor Asian Lady Beetle (Harmonia axyridis) on wine quality /

The Impact of the Multicolor Asian Lady Beetle (Harmonia axyridis) on Niagara Wine Quality The possible influence of Harmonia axyridis (the Multicolored Asian Lady Beetle) on the sensory properties of wine was investigated. H. axyridis beetles were added to white and red grape musts at a rate of 0, 1 or 10 per L, and a trained panel evaluated the finished wines using flavor-profiling techniques. Significant modification of both wine aroma and flavor characteristics were observed in the 10 beetlelL treatments, with smaller effects noted at the 1 beetlelL rate. Vinification in the presence of H. axyridis gave higher intensity scores for peanut, bell pepper and asparagus aromas and flavors in the white wines, and peanut, asparagus/bell pepper, and earthy/herbaceous aromas and flavors in the red wines. In addition, sweet, acid and bitter tastes were affected in red wines, and a general trend of decreasing fruit and floral intensities with increasing beetle rate was observed in both white and red wines. 15 ngIL Isopropylmethoxypyrazine was added to control wines and sensory profiles similar to high beetle treatments were obtained, supporting the hypothesis that methoxypyrazines from beetles are implicated in the taint. A trained panel evaluated the treated wines after 10 months of aging using the same sensory methods described above. Sensory profiles were very similar. Fennenting in the presence of Harmonia Axyridis (HA) had little influence on the chemical composition of the ftnished wine. The notable exception IS Isopropylmethoxypyrazine content, which was assessed usmg GC-MS analysis and showed increased concentration with increasing beetle nwnber for both white and red wmes. The influence of potential remedial treatments on the sensory properties of white and red wines tainted by Harmonia axyridis were also investigated. Bentonite, activated charcoal, oak chips, de-odorized oak chips, and UV or light irradiation were applied to tainted wine, and these wines evaluated chemically and sensorially. Both white and red wines treated with oak chips had strong oak characteristics, which masked the Harmonia axyridis-associated aroma and flavour attributes. In red wine, asparagus/bell pepper characteristics were decreased by bentonite and charcoal treatments. Only activated charcoal significantly decreased methoxypyrazine levels and only in white wine.