• Quantification of gamma-secretase activity in an endogenous context reveals biphasic GSI-mediated Notch/APP selectivity switch and the novel detection of potential proteolytic cleavage fragments of the Notch Intracellular Domain

      Mueller, Matthew Craig; Department of Biological Sciences
      Gamma-secretase is a promiscuous intra-membrane protease implicated in the proteolytic processing of two notable substrates: amyloid-precursor-protein (APP), in which gamma-secretase will irreversibly cleave to produce Amyloid-beta (Aβ) in Alzheimer’s disease; and the Notch receptor, where gamma-secretase is essential for liberating the Notch intracellular domain (NICD) to activate Notch-mediated transcriptional regulation in the nucleus. Gamma-secretase inhibitors such as DAPT and Avagacestat have been tested as therapies to prevent the formation of amyloid plaques, however, off-target interference with the Notch signalling pathway leading to Notch-related malignant side effects in clinical trials makes these pharmaceuticals unsuitable. The high-throughput search for selective drugs that block the production of Aβ but don’t interfere with the Notch signalling pathway has been hindered by a lack of reliability in detecting Notch signal inhibition in pre-clinical, cell-based assays with ectopic substrate expression. Therefore, the development of a highly-sensitive, high-throughput cell-based assay to quantify the level of proteolytic processing of APP and Notch by gamma-secretase is a promising addition to the gamma-secretase inhibitor drug discovery pipeline. This thesis presents the combination of immunofluorescence staining, western blotting, and the bromo-deoxyuridine (BrdU) cell proliferation assay as three orthogonal methods to sensitively quantify the gamma-secretase cleavage of Notch and APP in SH-SY5Y human neuroblastoma cells, which rely on active Notch signalling to maintain proliferation. Using our assay, we found that the selectivity for gamma-secretase to cleave APP versus Notch was dependent on the time the GSI was replenished before harvesting, which may directly reflect a GSI concentration-dependent selective potency. Using this high-throughput cell-based assay, cleavage of APP and Notch activation by gamma-secretase was sensitively quantified; while a novel profile of cell-type specific proteolytic fragments of the Notch ICD have been identified that may have biological implications in normal development and the pathological proliferation and metastasis of some cancers.
    • Radiation-induced changes in the export of photoassimilated carbon /|nBarry Jess Shelp. -- 260 0 St. Catharines, [Ont. : s. n.],

      Shelp, Barry Jess.; Department of Biological Sciences (Brock University, 1977-07-09)
      Low levels of ionizing radiation induce two translocation responses in soybean: a reduction in photoassimilate export from leaves and a change in the distribution pattern of exported photoassimilate within the plant. In this investigation these responses have been further studied specifically to ascertain the site of radiation damage and to better understand the physiological responses observed. Experimentally the primary data was obtained from studies in which a mature trifoliate leaf of a young soybean plant (Glycine ~ L. cultivar Harosoy '63) is isolated in a closed transparent chamber and allowed to photoassimilate 14C02 for 15 minutes. This is followed by an additional 45 ~_il'1;ute period before the plant is sectl.o ne d an d 14 C-ra dl' oactl.v.l ty d eterml. ne d'l n a 11 parts. Such 14c data provides one with the magnitude and distribution pattern of translocation. Further analyses were conducted to determine the relative levels of the major photosynthetic products using the techniques of paper chromatography and autoradiography. Since differences between control and irradiated P 1 ants were not 0 b serve d l' n t h e par tl't"lo nlng 0 f 14 C between the 80% ethanol-soluble and -insoluble fractions 14 or in the relative amounts of C-products of photosynthesis, the reduction in export in irradiated plants is not likely due to reduced availability of translocatable materials. Data presented in this thesis shows that photoassimilate export was not affected by gamma radiation until a threshold dose between 2.0 and 3.0 krads was reached. It was also observed that radiation-induced damage to the export process was capable of recovery in a period of 1 to 2 hours provided high light intensity was supplied. In contrast, the distribution pattern was shown to be extremely radiosensitive with a low threshold dose between .25 and .49 krads. Although this process was also capable of recovery,lt" occurred much earlier and was followed by a secondary effect which lasted at least for the duration of the experiments. The data presented in this thesis is interpreted to suggest that the sites of radiation action for the two translocation responses are different. In regards to photoassimilate export, the site of action of ionizing radiation is the leaf, quite possibly the process of photophosphorylation which may provide energy directly for phloem loading and for membrane integrity of the phloem tissue* In regards to the pattern of distribution of exported photoassimilate, the site is likely the apical sink, possibly the result of changes of levels of endogenous hormones. By the selection of radiation exposure dose and time post-irradiation, it is possible to affect independently these two processes suggesting that each may be regulated independent of the other and involves a distinct site.
    • Rapid [Gamma]-amino acid synthesis and the inhibition of the growth and development of oblique banded leaf-roller larvae

      Ramputh, Al-Idrissi.; Department of Biological Sciences (Brock University, 1996-07-09)
      The hypothesis that rapid y-aminobutyric acid (GABA) accumulation is a plant defense against phytophagous insects was investigated. Simulation of mechanical damage resulting from phytophagous insect activity increased soybean (Glycine max L.) leaf GABA 10- to 25-fold within 1 to 4 min. Pulverizing leaf tissue resulted in a value of 2. 15 (±O. 11 SE) ~mol GABA per gram fresh weight. Increasing the GABA levels in a synthetic diet from 1.6 to 2.6 Jlffiol GABA per gram fresh weight reduced the growth rates, developmental rates, total biomass (50% reduction), and survival rates (30% reduction) of cultured Oblique banded leaf-roller (OBLR) (Choristonellra rosacealla Harris) larvae. In field experiments OBLR larvae were found predominantly on young terminal leaves which have a reduced capacity to produce GABA in response to mechanical damage. Glutamate decarboxylase (GAD) is a cytosolic enzyme which catalyses the decarboxylation of L-Glu to GABA. GAD is a calmodulin binding enzyme whose activity is stimulated dramatically by increased cytosolic H+ or Ca2 + ion concentrations. Phytophagous insect activity will disrupt the cellular compartmentation of H+ and Ca2 +, activate GAD and subsequent GABA accumulation. In animals GABA is a major inhibitory neurotransmitter. The possible mechanisms resulting in GABA inhibited growth and development of insects are discussed.
    • Reactivity characteristics of cytochrome c, cytochrome oxidase and the electrostatic cytochrome c - cytochrome oxidase complex /

      Hill, Bruce.; Department of Biological Sciences (Brock University, 1979-06-09)
      The mechanism whereby cytochrome £ oxidase catalyses elec-. tron transfer from cytochrome £ to oxygen remains an unsolved problem. Polarographic and spectrophotometric activity measurements of purified, particulate and soluble forms of beef heart mitochondrial cytochrome c oxidase presented in this thesis confirm the following characteristics of the steady-state kinetics with respect to cytochrome £: (1) oxidation of ferrocytochrome c is first order under all conditions. -(2) The relationship between sustrate concentration and velocity is of the Michaelis-Menten type over a limited range of substrate. concentrations at high ionic strength. (3) ~he reaction rate is independent from oxygen concentration until very low levels of oxygen. (4) "Biphasic" kinetic plots of enzyme activity as a function of substrate concentration are found when the range of cytochrome c concentrations is extended; the biphasicity ~ is more apparent in low ionic strength buffer. These results imply two binding sites for cytochrome £ on the oxidase; one of high affinity and one of low affinity with Km values of 1.0 pM and 3.0 pM, respectively, under low ionic strength conditions. (5) Inhibition of the enzymic rate by azide is non-c~mpetitive with respect to cytochrome £ under all conditions indicating an internal electron transfer step, and not binding or dissociation of £ from the enzyme is rate limiting. The "tight" binding of cytochrome '£ to cytochrome c oxidase is confirmed in column chromatographic experiments. The complex has a cytochrome £:oxidase ratio of 1.0 and is dissociated in media of high ionic strength. Stopped-flow spectrophotometric studies of the reduction of equimolar mixtures and complexes of cytochrome c and the oxidase were initiated in an attempt to assess the functional relevance of such a complex. Two alternative routes -for reduction of the oxidase, under conditions where the predominant species is the £ - aa3 complex, are postulated; (i) electron transfer via tightly bound cytochrome £, (ii) electron transfer via a small population of free cytochrome c interacting at the "loose" binding site implied from kinetic studies. It is impossible to conclude, based on the results obtained, which path is responsible for the reduction of cytochrome a. The rate of reduction by various reductants of free cytochrome £ in high and low ionic strength and of cytochrome £ electrostatically bound to cytochrome oxidase was investigated. Ascorbate, a negatively charged reagent, reduces free cytochrome £ with a rate constant dependent on ionic strength, whereas neutral reagents TMPD and DAD were relatively unaffected by ionic strength in their reduction of cytochrome c. The zwitterion cysteine behaved similarly to uncharged reductants DAD and TI~PD in exhibiting only a marginal response to ionic strength. Ascorbate reduces bound cytochrome £ only slowly, but DAD and TMPD reduce bound cytochrome £ rapidly. Reduction of cytochrome £ by DAD and TMPD in the £ - aa3 complex was enhanced lO-fold over DAD reduction of free £ and 4-fold over TMPD reduction of free c. Thus, the importance of ionic strength on the reactivity of cytochrome £ was observed with the general conclusion being that on the cytochrome £ molecule areas for anion (ie. phosphate) binding, ascorbate reduction and complexation to the oxidase overlap. The increased reducibility for bound cytochrome £ by reductants DAD and TMPD supports a suggested conformational change of electrostatically bound c compare.d to free .£. In addition, analysis of electron distribution between cytochromes £ and a in the complex suggest that the midpotential of cytochrome ~ changes with the redox state of the oxidase. Such evidence supports models of the oxidase which suggest interactions within the enzyme (or c - enzyme complex) result in altered midpoint potentials of the redox centers.
    • The regulation of oat coleoptile phosphoenolpyruvate carboxylase and malic enzyme by Hâ ½ and metabolites : kinetic evidence for and against a cytosolic pH-stat

      Smith, Ceredwyn Elizabeth.; Department of Biological Sciences (Brock University, 1979-07-09)
      Phosphoenolpyruvate carboxylase (PEPC) and malic enzyme activities in soluble protein extracts of Avena coleoptiles were investigated to determine whether their kinetics were consistent with a role in cytosol pH regulation. Malic enzyme activity was specific for NADP+ and Mn2+. Maximal labelled product formation from [14C]-substrates required the presence of all coenzymes, cofactors and substrates. Plots of rate versus malate concentration, and linear transformations there- 2 of, indicated typical Michaelis-Menten kinetics at non-saturating malate levels and substrate inhibition at higher malate levels. pH increases between 6.5 and 7.25 increased near-optimal activity, decreased the degree of substrate inhibition and the Kmapp(Mn2+) but did not affect the Vmax or Kmapp(malate). Transformed data of PEPC activity demonstrated non-linear plots indicative of non-Michaelian kinetics. pH increases between 7.0 and 7.6 increased the Vmax and decreased the Km app (Mg2+) but did not affect the Kmapp(PEP). Various carboxylic acids and phosphorylated sugars inhibited PEPC and malic enzyme activities, and these effects decreased with pH increases. Metabolite inhibited malic enzyme activity was non-competitive and resulted mainly from Mn2+ chelation. In contrast, metabolite inhibited PEPC activity was unique for each compound tested, being variously dependent on the PEP concentration and the pH employed. These results indicate that fluctuations in pH and metabolite levels affect PEPC and malic enzyme activities similarly and that 3 the in vitro properties of PEPC are consistent with its proposed role in a pH-stat, whereas the in vitro properties of the malic enzyme cannot be interpreted in terms of a role in pH regulation.
    • Relationship between photoperiod, plasma concentration of ionic calcium and the histology of the prolactin-secreting cells of the rostral pars distalis of the pituitary gland, the corpuscles of stannius and the ultimobranchial gland in the goldfish, Carassius auratus L. /

      Calarco, Ann Marie.; Department of Biological Sciences (Brock University, 1980-06-09)
      The relationship between photoperiod, plasma concentration of ionic calcium and the histology of the prolactin-secreting cells of the rostral pars distalis of the pituitary gland, the Corpuscles of Stannius and the Ultimobranchial gland were investigated. Neither the plasma concentration of ionic calcium nor histologically apparent prolactin cell activity could be correlated with photoperiod. Some evidence of a photoperiodic effect on both the Corpuscles of Stannius and the Ultimobranchial gland was obtained. The expected reciprocal relationship between the activity of these glands was not obvious at the histological level . Quantitative and qualitative analysis at the light microscope level revealed, however, that the hormone prolactin-secreting eta cells of the rostral pars distalis and the hypocalcin-secreting cells of the Corpuscles of Stannius may be arranged in a lamellar pattern comprized of synchronous bands of cells in like-phase of a secretory cycle consisting of four stages - synthesis, storage, release and reorganization. Such synchronized cell cycles in these glands have not heretofore been described in literature. It is suggested that the maintenance of at least 255? of the cells in any one phase of the cycle ensures a supply of the required hormone at all times.
    • A relationship between photosynthesis and translocation in plants stressed by ionizing radiation

      McCabe, James B.; Department of Biological Sciences (Brock University, 1977-07-09)
      Since previous investigations have shown that low levels of ionizing radiation can induce a reduction in the rates of apparent photosynthesis and in the magnitude of photoassimilated l4C exported out of a leaf, the present studies were designed and conducted to determine the relationship, if any, between the radiation effects on these two physiological processes. The experiments were particularly designed to determine if the radiation-induced reduction in export is the result of the reduction in photosynthesis and hence availability of materials for translocation or the result of a reduction in the amount of energy available for the vein loading process. This study has shown that the radiation-induced reduction in l4C export out of a leaf is likely related to a loss of energy available for the vein loading process rather than a reduction in the supply of materials available for export due to reduced C02 uptake. The process of photophosphorylation was shown to be reduced by exposure to radiation to an extent similar to the reduction in the export of l4C which was also observed. Both of these processes returned to their pre-irradiation rates 120 minutes following radiatruon exposure. The rate of photosynthetic C02 uptake was also reduced by radiation exposur~ howeve~ this process did not return to the control level nor was the extent of reduction as large as observed for photophosphorylation and photoassimilate export. The observed relationship between the reductions of export and photoph~sphorylation pointed to the utilization of photosynthetically produced ATP in the vein loading process. The radiation-induced reduction in the export of l4C was observed at the highest light intensity used in this study which would also imply the involvement of the photophosphorylation process as an energy seurce for vein loading. The lack of radiation-induced reduction in export at low light intensities was interpreted as being due to the utilization of respiratory derived ATP, a process known to be insensitive to radiation at the levels used in this study, as the energy source for the vein loading process. Studies using plants not stressed by radiation showed that there was an increase in export of 14C with higher light intensities. In summary, the data has been interpreted as showing that at high light intensities the ATP, produced by photophosphorylation, is available for use in the vein loading process. The site of ATP utilization could not be determined from the data obtained in this study but possible sites have been indicated from the work done by other physiologists and are discussed in the thesis.
    • The relationship between stem-form, stand-closure and site -conditions : the influence of environmental conditions on tree allometry and forest structure in west-central Alberta

      Jaques, Kathleen E.; Department of Biological Sciences (Brock University, 1995-07-09)
      Changes in the configuration of a tree stern result insignificant differences in its total volume and in the proportion of that volume that is merchantable timber. Tree allometry, as represented by stem-fo~, is the result of the vertical force of gravity and the horizontal force of wind. The effect of wind force is demonstrated in the relationship between stem-form, standclosure and site-conditions. An increase in wind force on the individual tree due to a decrease in stand density should produce a more tapered tree. The density of the stand is determined by the conditions that the trees are growing under. The ability of the tree to respond to increased wind force may also be a function of these conditions . This stem-form/stand-closure/site-conditions relationship was examined using a pre-existing database from westcentral Alberta. This database consisted of environmental, vegetation, soils and timber data covering a wide range of sites. There were 653 sample trees with 82 variables that formed the basis of the analysis. There were eight tree species consisting of Pinus contorta, Picea mariana, Picea engelmannii x glauca, Abies lasiocarpa, Larix laricina, Populus tremuloides, Betula papyrifera and Populus balsamifera plus a comprehensive all-species data set. As the actual conformation of the stern is very individual, stem-fo~was represented by the diameter at breast height to total height r~tio. The four stand-closure variables, crown closure, total basal area, total volume and total number of stems were reduced to total basal area and total number of stems utilizing a bivariate correlation matrix by species. Site-conditions were subdivided into macro, meso and micro variables and reduced in number 3 using cross-tabulations, bivariate correlation and principal components analysis as screening tools. The stem-fo~/stand-closure relationship was examined using bivariate correlation coefficients for stem-fo~ with total number of stems and stem-fo~ with total basal area. The stem-fo~/site-conditions and the stand-closure/site- conditions relationships were examined using multiple correlation coefficients. The stem-form/stand-closure/site-conditions relationship was examined using multiple correlation coefficients in separate analyses for both total number of stems and total basal area. An increase in stand-closure produced a decrease in stem-form for both total number of stems and total basal area for most species. There was a significant relationship between stem-form and site-conditions and between stand-closure and site-conditions for both total number of stems and total basal area for most species. There was a significant relationship between the stemform and site-conditions, including the stand-closure, for most species; total number of stems was involved independently of the site-conditions in the prediction of stem-form and total basal area was not. Larix laricina and Betula papyrifera were the exceptions to the trends observed with most species. The influence of both stand-closure (total number of stems in particular) and site-conditions (elevation in particular) suggest that forest management practices should include these- ecological parameters in determining appropriate restocking levels.
    • Reproduction in the temperate crayfish, Orconectes rusticus: intermale aggression, copulatary behaviour, and sperm competition

      Snedden, William Andrew.; Department of Biological Sciences (Brock University, 1988-07-09)
      The chelipeds of Orconectes rusticus are sexually dimorphic; males possessing the larger. Males use their chelae in intermale aggressive interactions, both to threaten, and assault opponents. In dyadic interactions males with larger chelae were dominant over otherwise physically similar opponents. A high frequency of attack behaviour, coupled with a low frequency of threats during these interactions indicates that actual physical contact is required for opponent assessment. Large clawed males oriented females into the copulatory position faster than small clawed males. Females more frequently escaped the precopulatory-grasp attempts of small clawed males. Additionally, male-female pairs that included a large clawed male remained in copula longer than pairs that included a small clawed male. Sperm of the second male to mate took precedence over the sperm of the primary male. Sperm precedence was incomplete; about 900/0 paternity accrued to the second male.
    • Reproductive behaviour of male Xylocopa virginica and the influence of body size, nestmates, and siblings on territory defence

      Duff, Lyndon; Department of Biological Sciences
      Males of Xylocopa virginica are territorial like many other bee species. Males interact aggressively to displace other males from territories. Body size is known to influence resource holding potential in many other taxa, and studies of bees suggest that body size is important for territorial males. Familiarity and the avoidance of kin competition are also known to influence territorial behaviours in other taxa but has not been studied in male bees. Recent evidence suggests that nestmate recognition occurs in X. virginica and there is also evidence for the avoidance of kin competition. This thesis tests whether body size, familiarity, and kinship influence territorial interactions using social networking tools. Around half of all males attempted to establish or defend a territory. Males that established or defended territories are larger than males that did not. Male body size has a weak positive influence on hover rates, related to holding territories, the number of hovering neighbours each male had, and the number of males each male chased or fled in defence of territories. I found no evidence to support that familiarity influences aggressive behaviours, but there is a strong correlation with the number of neighbours a male had and the number of males it chased or fled. Brothers estimated from microsatellite genotypes are no more aggressive to each other than to non-siblings. However, the results indicate that several sets of brothers overwintered in different nests, which does not coincide with the behavioural patterns described in the literature. This study is the first step towards understanding the influence of familiarity and kin competition on male behaviour in a taxonomic group with a wide array of mate-locating strategies. Discussed herein are the importance of continued research on mating systems and mate-locating strategies of bees, as well as outlining future projects to address several gaps in knowledge that remain after this study.
    • Response rainbow trout (Salmon gairdneri) blood gas transport system to temperature, oxygen availability and photoperiod

      Tun, Nini.; Department of Biological Sciences (Brock University, 1984-07-09)
      Hematological status in rainbow trout, Salmo gairdneri, was examined in relation to eight combinations of three environmental fa ctors; temperature (5°, 20°C), oxygen availability «35%, >70% saturation) and photoperiod (16L:8D, 8L:16D) and evaluated by 3-factor analysis of variance. Hemog l obin and hematocrit , indicators of oxygenc arrying capacity increased significantly at the higher temperature, following exposure to hypoxia and in relation to reduced light period. Significant variations in mean corpuscular hemoglobin concentration were not detected. The effects of temperature and oxygen availability were more pronounced than that of photoperiod which was generally masked. Although oxygen availability and photoperiod did not interact with temperature, the interaction of the former fac tors was significant. Elec trophoresis revealed twelve hemoglobin isomorphs. Relative concentration changes were found in re lation to the factors c onsidered with temperature>hypoxia>photoperiod. Howeve r , in terms of absolute concentration, effects were hypoxia>temperature>photoperiod. Photoperiod effects were again masked by temperature and (or) hypoxia. Red cell +2 l eve ls of [CI ] and [Mg ], critical elements in the hemoglobin-oxygen affinity regulating system, were also significantly altered. Red cell CI +2 was influenced only by temperature ; Mg by temper ature and oxygen. No photoperiod influence on either ions was observed. Under nominal 'summer' conditions, these changes point to the likelihood of increases in oxygen-c arrying c apac ity coupled with low Hb-02 affinity adjustments which would be expected to increase oxygen delivery rates to their more rapidly metabolising tissues.
    • Responses of crayfish to a reflective environment /

      May, Holly Y.; Department of Biological Sciences (Brock University, 2007-06-04)
      This thesis examines how dominance status of crayfish alters responses to their own reflection. Crayfish are social animals that fight to develop a dominance hierarchy consisting of dominant and subordinate members. After socialization, crayfish were videotaped in an aquarium with mirrors on one half of the tank and a non-reflective plastic on the other half. Dominants paired for 14 days perform more cornering, turning, crossing and spent more time in a reflective environment versus a non-reflective environment. Subordinate crayfish exhibit more reverse walking in a mirrored environment while isolated crayfish show no preference for reflection. This change in behaviour occurs immediately for dominants paired for 30 min while subordinates require 3 days of pairing to exhibit the same behaviour as subordinate crayfish paired for 14 days. Thus, 30 min of pairing is required to enhance responses to a reflection observed in dominant crayfish while 3 days is required to decrease subordinate responses to a reflection. These findings propose that male socialized crayfish respond to their mirror image as they do a male conspecific. Their responses depend on both their dominance status and the length of socialization which suggests that crayfish are learning to behave in a characteristic manner as a result of their social experience.
    • Restoration and succession of a bee community in southern St. Catharines, Ontario, within a ten-year study period

      Onuferko, Thomas; Department of Biological Sciences (Brock University, 2014-01-10)
      A wild bee community in southern St. Catharines, Ontario, Canada, was studied from 2003 to 2012 to analyze the effects of primary succession on abundance and diversity. At a former landfill site near Brock University, which previously contained no bees, the number of bees and bee species was expected to increase rapidly following measures to restore the site to grassy meadow habitat. The Intermediate Disturbance Hypothesis (IDH) states that over time, succession occurs. Abundance and diversity increase initially and peak when pioneers coexist with specialized species, then decline because of competitive exclusion. Alternatively, abundance and diversity may continue to increase and stabilize without declining. Bees were sampled repeatedly among years from newer restoration sites (revegetated in 2003), older restoration sites on the periphery of the former landfill (revegetated in 2000), and nearby low disturbance grassy field (i.e. control) sites. In the newer sites, bee abundance and diversity increased then decreased while in older restoration and control sites mainly decreased. This pattern of succession matches the general predictions of the IDH, although declines were at least partially related to drought. By 2006, total bee abundance levels converged among all sites, indicating rapid colonization and succession, and by 2012 diversity levels were similar among sites as well, suggesting that the bee community was fully restored or nearly so within the ten-year study period.
    • Retinoic Acid and the Underlying Cellular Mechanisms involved in Neurite Outgrowth and Growth Cone Turning during Regeneration

      Nasser, Tamara Israa-Nadine; Department of Biological Sciences
      The involvement of retinoic acid (RA) in nervous system regeneration has been well documented, though the precise cellular and molecular mechanisms have not yet been fully determined. During regeneration, RA can exert trophic support for cells, as well as tropic effects to guide neurite outgrowth. Cultured neurons of the Lymnaea central nervous system (CNS) have previously been used to investigate the role of RA in neurite outgrowth and growth cone guidance. Recently however, a novel phenomenon has been identified, in which neurite outgrowth occurs from cut nerves of Lymnaea CNS “floating” on the surface of cultured medium. In this study, I examined whether RA could induce or guide neurite outgrowth from this novel preparation. Unlike previous findings with cultured neurons, there was a lack of consistent effects of 9-cis and all-trans RA in promoting neurite outgrowth from the floating CNS. However, the growth cones of these floating neurites were found to turn toward a local source of RA, indicating for the first time, that they are capable of responding to guidance cues, even in the absence of adhesion to a solid substrate. The cellular mechanisms of RA-induced growth cone turning were then conducted on cultured neurons. I demonstrated that various retinoid receptor agonists could mimic the chemotropic effects of RA, providing further evidence for a potential role of retinoid X receptor and retinoic acid receptors in growth cone turning. I also examined potential downstream effectors involved in this chemotropic response to RA and provided the first evidence that the intracellular signaling pathway likely involves the Rho GTPase, Rac. However, it was also shown that the involvement of Rac in mediating the RA-induced growth cone turning differed depending on whether the turning was induced by endogenous or synthetic retinoids, and also whether the growth cones were still attached to the cell body or not. Overall, these data provide new insights into the mechanisms by which retinoids affect neurite outgrowth and growth cone behaviour during regeneration of the nervous system.
    • The role of cyclic nucleotides in modulation of crayfish neuromuscular junctions by a neuropeptide /

      Badhwar, Amit.; Department of Biological Sciences (Brock University, 1999-05-21)
      DF2, a heptapeptide, is a member of the family of FMRFamide-like peptides and has been shown to increase the amount of transmitter released at neuromuscular junctions of the crayfish, Procambarus clarkit Recent evidence has shown that protein kinase C (PKC), calcium/calmodulin-dependent protein kinase II (CaMKII) and the cAMPdependent protein kinase (PKA) play a role in the neuromodulatory pathway of DF2. The involvement of these kinases led to the prediction that a G-protein-coupled receptor (GPCR) is activated by DF2 due to the role that each kinase plays in traditional GPCR pathways seen in other organisms and in other cells. G-proteins can also act on an enzyme that generates cyclic guanosine monophosphate (cGMP) which mediates its effects through a cGMP-dependent protein kinase (PKG). This thesis addresses the question of whether or not DF2's effects on synaptic transmission in crayfish are mediated by the cyclic nucleotides cAMP and cGMP. The effects of DF2 on synaptic transmission were examined using deep abdominal extensor muscles of the crayfish Procambarus clarkii. An identified motor neuron was stimulated, and excitatory post-synaptic potentials (EPSPs) were recorded in abdominal extensor muscle LI . A number of activators and inhibitors were used to determine whether or not cAMP, PKA, cGMP and PKG mediate the effect of this peptide. Chemicals that are known to activate PKA (Sp-cAMPS) and/or PKG (8-pCPTcGMP) mimic and potentiate DF2's effect by increasing EPSP amplitude. Inhibitors of either PKA (Rp-cAMPS) or PKG (Rp-8-pCPT-cGMPS) block a portion of the increase in EPSP amplitude induced by the peptide. When both kinase inhibitors are applied simultaneously, the entire effect of DF2 on EPSPs is blocked. The PKG inhibitor blocks the effects of a PKG activator but does not alter the effect of a PKA activator on EPSP amplitude. Thus, the PKG inhibitor appears to be relatively specific for PKG. A trend in the data suggests that the PKA inhibitor blocks a portion of the response elicited by the PKG activator. Thus, the PKA inhibitor may be less specific for PKA. Phosphodiesterase inhibitors, which are known to inhibit the breakdown of cAMP (IBMX) and/or cGMP (mdBAMQ), potentiate the effect of the peptide. These results support the hypothesis that cAMP and cGMP, acting through their respective protein kinase enzymes, mediate the ability of DFi to increase transmitter output.
    • Role of Exopolysaccharide in Pantoea agglomerans Interactions with Bacteriophages

      Zaprzala, Patrick; Department of Biological Sciences
      Fire blight is the common name given to a disease caused by phytopathogen, Erwinia amylovora. This pathogen infects plants belonging to the rosaceous family, particularly the commercially grown apple and pear cultivars in North America. Agriculture and Agri-Food Canada Research team has focused on the use of bacteriophages as biological control agents for the control of fire blight. Bacteriophage persistence in the orchard environment can be affected by various environmental factors such as exposure to ultraviolet light and dry conditions. In order to improve phage viability during application, a carrier-phage system was developed that uses a non-pathogenic epiphytic bacterium, named “the carrier”, that protects the bacteriophages during processing and field applications. The bacterium used is Pantoea agglomerans, which delivers and propagates the phages to the open blossoms, prior to the arrival of E. amylovora. In addition, the carrier alone may act as a biological control agent on the blossom surface, competing for space and producing antibiotics targeting E. amylovora. The aim of this study was study the interaction between the phage and the carrier bacterium, P. agglomerans. The exopolysaccharide (EPS) layer, is the first cellular component which bacteriophages encounter during the infection of P. agglomerans. PCR was conducted and it confirmed that 6 genes involved in EPS biosynthesis were present within the four different isolates of P. agglomerans. A CRISPR/Cas9 knockout and lambda red recombineering protocol was used to construct EPS deficient P. agglomerans mutants. The deficient populations were confirmed using colony appearance, PCR and Sangar sequencing. A quantitative PCR was conducted to quantify and compare the different bacteriophage populations between the wild-type and deficient EPS mutants. The EPS mutants were found to have shown a significantly decreased or complete lack of growth in both Myoviridae and Podoviridae infections.
    • Role of Exopolysaccharides and Monosaccharides in Erwinia amylovora Resistance to Bacteriophages

      Sjaarda, David; Department of Biological Sciences (Brock University, 2012-08-09)
      Fire blight is a disease caused by the phytopathogenic bacterium Erwinia amylovora, an economically important pathogen in the commercial production of apples and pears. Bacteriophages have been proposed as a commercial biopesticide to relieve the pressures on apple and pear production and provide alternatives to existing biological control options. This work reports on the investigation of host resistance in the development of a phage biopesticide. Exopolysaccharide (EPS) deficient bacterial mutants were generated through recombineering to investigate the role of EPS in bacteriophage adsorption and infection. The mutants that were deficient in amylovoran production were avirulent and resistant to infection by phages of the Podoviridae and some of the Siphoviridae family. Levan deficient bacterial mutants resulted in reduced phage titers in some phages from the Myoviridae family. Exopolysaccharide mimetic monosaccharides were used to demonstrate that levan and amylovoran play an important role in phage attack of E. amylovora.
    • The role of water in determining structure and function of macromolecules and macromolecular assemblies /

      Fuller, Nola L.; Department of Biological Sciences (Brock University, 2002-07-14)
      The interaction of biological molecules with water is an important determinant of structural properties both in molecular assemblies, and in conformation of individual macromolecules. By observing the effects of manipulating the activity of water (which can be accomplished by limiting its concentration or by adding additional solutes, "osmotic stress"), one can learn something about intrinsic physical properties of biological molecules as well as measure an energetic contribution of closely associated water molecules to overall equilibria in biological reactions. Here two such studies are reported. The first of these examines several species of lysolipid which, while present in relatively low concentrations in biomembranes, have been shown to affect many cellular processes involving membrane-protein or membrane-membrane interactions. Monolayer elastic constants were determined by combining X-ray diffraction and the osmotic stress technique. Spontaneous radii of curvature of lysophosphatidylcholines were determined to be positive and in the range +30A to +70A, while lysophosphatidylethanolamines proved to be essentially flat. Neither lysolipid significantly affected the bending modulus of the monolayer in which it was incorporated. The second study examines the role of water in theprocess of polymerization of actin into filaments. Water activity was manipulated by adding osmolytes and the effect on the equilibrium dissociation constant (measured as the criticalmonomer concentration) was determined. As water activity was decreased, the critical concentration was reduced for Ca-actin but not for Mg-actin, suggesting that 10-12 fewer water molecules are associated with Ca-actin in the polymerized state. Thisunexpectedly small amount of water is discussed in the context of the common structural motif of a nucleotide binding cleft.