Investigation of the mechanism of transfer of a-tocopherol by the human a-tocopherol transfer protein (H-a-TTP) /

The human a-tocopherol transfer protein (h-a-TTP) is understood to be the entity responsible for the specific retention of a-tocopherol (a-toc) in human tissues over all other forms of vitamin E obtained from the diet. a-Tocopherol is the most biologically active form of vitamin E, and to date has been studied extensively with regard to its antioxidant properties and its role of terminating membrane lipid peroxidation chain reactions. However, information surrounding the distribution of a-tocopherol, specifically its delivery to intracellular membranes by a-TTP, is still unclear and the molecular factors influencing transfer remain elusive. To investigate the mechanism of ligand transfer by the h-a-TTP, a fluorescent analogue of a-toc has been used in the development of a fluorescence resonance energy transfer (FRET) assay. (/?)-2,5,7,8-tetramethyl-2-[9-(7-nitro-benzo[l,2,5]oxdiazol-4-ylamino)-nonyl]- chroman-6-ol (NBD-toc) has allowed for the development of the FRET-based ligand transfer assay. This ligand has been utilized in a series of experiments where changes were made to acceptor lipid membrane concentration and composition, as well as to the ionic strength and viscosity of the buffer medium. Such changes have yielded evidence supporting a collisional mechanism of ligand transfer by a-TTP, and have brought to light a new line of inquiry pertaining to the nature of the forces governing the collisional transfer interaction. Through elucidation of the transfer mechanism type, a deeper understanding of the transfer event and the in vivo fate of a-tocopherol have been obtained. Furthermore, the results presented here allow for a deeper investigation of the forces controlling the collisional protein-membrane interaction and their effect on the transfer of a-toc to membranes. Future investigation in this direction will raise the possibility of a complete understanding of the molecular events surrounding the distribution of a-toc within the cell and to the body's tissues.

Optical properties of organic superconductor K-(BETS)2FeBr4 /

K-(BETS)2FeBr4 is a quasi-2D charge transfer organic metal with interesting electronic and magnetic properties. It undergoes a transition to an antiferromagnetic (AF) state at ambient pressure at the Neel temperature (T^^) = 2.5 K, as well as to a superconducting (SC) state at 1.1 K [1]. The temperature dependence of the electrical resistivity shows a small decrease at T;v indicating the resistivity drops as a result of the onset of the ordering of Fe'*''" spins. A sharp drop in the resistivity at 1.1 K is due to its superconducting transition. The temperature dependence of the susceptibility indicates an antiferromagnetic spin structure with the easy axis parallel to the a-axis. The specific heat at zero-field shows a large peak at about 2.4 K, which corresponds to the antiferromagnetic transition temperature (Tat) and no anomaly is observed around the superconducting transition temperature (1.1 K) demonstrating that the magnetically ordered state is not destroyed by the appearance of another phase transition (the superconducting transition) in the 7r-electron layers [1], [2]. This work presents an investigation of how the low frequency electromagnetic response is affected by the antiferromagnetic and superconducting states, as well as the onset of strong correlation. The location of the easy axis of three samples was determined and polarized thermal reflectance measurements of these «-(BETS)2FeBr4 samples oriented with their vertical axis along the a- and c axes were then carried out using a *He refrigerator cryostat and a Martin-Puplett type polarizing interferometer at various temperatures (T = 0.5 K, 1.4 K. 1.9 K, 2.8 K) above and below the superconducting state and/or antiferromagnetic state. Comparison of the SC state to the normal state along the o- and c-axes indicates a rising thermal reflectance at low frequencies (below 10 cm"' ) which may be a manifestation of the superconducting energy gap. A dip-Hke feature is detected at low frequencies (below 15 cm"') in the thermal reflectance plots which probe the antiferromagnetic state along the two axes, and may be due to the opening of a gap in the excitation spectrum as a result of the antiferromagnetism. In another set of experiments, thermal reflectance measurements carried out along the a- and c-axes at higher temperatures (10 K-80 K) show that the reflectivity decreases with increasing temperature to 60 K (the coherence temperature) above which it increases again. Comparison of the thermal reflectance plots along the a- and c-axes at higher temperatures reveals an anisotropy between these two axes. The Hagen-Rubens thermal reflectance plots corresponding to an average over the ac-plane were calculated using experimental hterature resistivity values. Comparison of the Hagen-Rubens plots with the experimental thermal reflectance along the a- and c-axes indicates that both exhibit the general trend of a decrease in thermal reflectance with increasing frequency, however the calculated Hagen-Rubens thermal reflectance at different temperatures is much lower than the experimental curves.

Fimbriae of coprinus cinereus, schizophyllum commune and ustilago violacea = structural aspects and a role in conjugation

Surface fibrils (fimbriae) have been observed on fungi from every major group. Fimbriae are thought to be involved in the following cell to cell interactions: conjugation, flocculation and adhesion. Several higher fungi exibit two other types of interactions: hyphal fusion (anastomosis) and clamp connection formation. As a prelude to examining the role of fimbriae in these processes, the fimbriae of two fungi that undergo these fusion events were examined. Electron microscopy studies revealed that Coprinus cinereus and Schizophyllum commune are fimbriated. C. cinereus fimbriae were 5 nm in diameter and 0.5 to 20 11m in length. Fimbriae of C. cinereus oidia were more numerous and longer than those of the hyphal stage. S. commune fimbriae were also 5 nm in diameter, but were only 0.5 to 2 11m in length. There was an unequal distribution of fimbriae on the hyphal surfaces of S. commune . Fimbriae were sparsely distributed over the entire hyphal surface, with higher densities of fibrils present on the side growths of the hyphae found in the older sections of the mycelium. Antiserum raised against Ustilago violacea fimbrial protein (AU) crossreacted strongly with 37 and 39 kd C. cinereus mycelial proteins. In contrast, AU bound very weakly to 89 and 92 kd S. commune mycelial proteins. Since AU cross-reacted poorly with S. commune fimbrial proteins, it was impossible to further characterize the fimbriae of this specIes. The 37 and 39 kd C. cinereus proteins, were isolated by electroelution and were shown to be able to form fibrils the same diameter as oidial fimbriae. The 37 kd protein was shown to be composed of several proteins with isoelectric points ranging from pH 6.1 to 7.63. Furthermore, the 37 kd protein was found to be multimeric, while the 39 kd protein was not. These results strongly suggested that the 37 kd protein is the structural fimbrial protein of C. cine reus . Finally, a series of experiments were designed to determine whether fimbriae are required for conjugation in U. violacea Conjugation was inhibited significantly with AU, but not with pre-immune serum or AU preincubated with purified fimbrial protein. Thus, it was concluded that fimbriae play a central role in mating in this organism.

The determination of sinigrin in Brassica, and investigations into the use of allyl-isothiocyanate as a nematicide

The goal of this thesis was to study factors related to the development of Brassica juncea as a sustainable nematicide. Brassica juncea is characterized by the glycoside (glucosinolate) sinigrin. Various methods were developed for the determination of sinigrin in Brassica juncea tissue extracts. Sinigrin concentrations in plant tissues at various stages of growth were monitored. Sinigrin enzymatically breaks down into allylisothiocyanate (AITC). AITC is unstable in aqueous solution and degradation was studied in water and in soil. Finally, the toxicity of AITC against the root-lesion nematode (Pratylenchus penetrans) was determined. A method was developed to extract sinigrin from whole Brassica j uncea tissues. The optimal time of extraction wi th boiling phosphate buffer (0.7mM, pH=6.38) and methanol/water (70:30 v/v) solutions were both 25 minutes. Methanol/water extracted 13% greater amount of sinigrin than phosphate buffer solution. Degradation of sinigrin in boiling phosphate buffer solution (0.13%/minute) was similar to the loss of sinigrin during the extraction procedure. The loss of sinigrin from boiling methanol/water was estimated to be O.Ol%/minute. Brassica juncea extract clean up was accomplished by an ion-pair solid phase extraction (SPE) method. The recovery of sinigrin was 92.6% and coextractive impurities were not detected in the cleaned up extract. Several high performance liquid chromatography (HPLC) methods were developed for the determination of sinigrin. All the developed methods employed an isocratic mobile phase system wi th a low concentration of phosphate buffer solution, ammonium acetate solution or an ion-pair reagent solution. A step gradient system was also developed. The method involved preconditioning the analytical column with phosphate buffer solution and then switching the mobile phase to 100% water after sample injection.Sinigrin and benzyl-glucosinolate were both studied by HPLC particle beam negative chemical ionization mass spectrometry (HPLCPB- NCI-MS). Comparison of the mass spectra revealed the presence of fragments arising from the ~hioglucose moiety and glucosinolate side-chain. Variation in the slnlgrin concentration within Brassica juncea plants was studied (Domo and Cutlass cuItivars). The sinigrin concentration in the top three leaves was studied during growth of each cultivar. For Cutlass, the minimum (200~100~g/g) and maximum (1300~200~g/g) concentrations were observed at the third and seventh week after planting, respectively. For Domo, the minimum (190~70~g/g) and maximum (1100~400~g/g) concentrations were observed at the fourth and eighth week after planting, respectively. The highest sinigrin concentration was observed in flower tissues 2050±90~g/g and 2300±100~g/g for Cutlass and Domo cultivars, respectively. Physical properties of AITC were studied. The solubility of AITC in water was determined to be approximately 1290~g/ml at 24°C. An HPLC method was developed for the separation of degradation compounds from aqueous AITC sample solutions. Some of the degradation compounds identified have not been reported in the literature: allyl-thiourea, allyl-thiocyanate and diallyl-sulfide. In water, AITC degradation to' diallyl-thiourea was favored at basic pH (9.07) and degradation to diallyl-sulfide was favored at acidic pH (4 . 97). It wap necessary to amend the aqueous AITC sample solution with acetonitrile ?efore injection into the HPLC system. The acetonitrile amendment considerably improved AITC recovery and the reproducibility of the results. The half-life of aqueous AITC degradation at room temperature did not follow first-order kinetics. Beginning with a 1084~g/ml solution, the half-life was 633 hours. Wi th an ini tial AITC concentration of 335~g/ml the half-life was 865 hours. At 35°C the half-life AITC was 76+4 hours essentially independent of the iiisolution pH over the range of pH=4.97 to 9.07 (1000~g/ml). AITC degradation was also studied in soil at 35°C; after 24 hours approximately 75% of the initial AITC addition was unrecoverable by water extraction. The ECso of aqueous AITC against the root-lesion nematode (Pratylenchus penetrans) was determined to be approximately 20~g/ml at one hour exposure of the nematode to the test solution. The toxicological study was also performed with a myrosinase treated Brassica juncea extract. Myrosinase treatment of the Brassica juncea extract gave nearly quantitative conversion of sinigrin into AITC. The myrosinase treated extract was of the same efficacy as an aqueous AITC solution of equivalent concentration. The work of this thesis was focused upon understanding parameters relevant to the development of Brassica juncea as a sustainable nematicide. The broad range of experiments were undertaken in support of a research priority at Agriculture and Agri-Food Canada.

Pressure dependence of superconducting properties of MgB2 /

We prepared samples of MgB2 and ran sets of experiments aimed for investigation of superconducting properties under pressure. We found the value of pressure derivative of the transition temperature -1.2 ± 0.05 K/GPa. Then, using McMillan formula, we found that the main contribution to the change of the transition temperature under the pressure is due to the change in phonon frequencies. Griineisen parameter was calculated to be 7g = 2.4. Our results suggest that MgB2 is a conventional superconductor.

The role of the GABAergic system in the production of ultrasonic vocalization in rats /

Ultrasonic vocalization plays an important role in intraspecies communication for rats. It has been well demonstrated that rats will emit 22kHz vocalization in stressfiil or threatening situations. Although the neural mechanism underlying vocahzation is not well understood, it is known that chohnergic input to the basal forebrain induces such alarm calls. A number of experiments have found that intracerebral injection of carbachol, a predominantly muscarinic agonist, into die anterior hypothalamic/preoptic area (AH/POA) rehably induces vocalization similar to naturally emitted ultrasonic calls. It has also been shown that carbachol has extensive inhibitory effects on neuronal firing in the same area. This result impUes that the inhibitory effects of carbachol in the AH/POA could trigger vocahzation, and that the GABAergic system could be involved. The purpose of this study is to investigate the effects ofGABA agonists and antagonists on flie production of carbachol induced 22kHz vocalization. The following hypotheses were examined: 1) apphcation ofGABA (a naturally occurring inhibitory neurotransmitter) will have a synergistic effect with carbachol, increasing vocalization; and 2) tiie apphcation ofGABA antagonists (picrotoxin or bicuculline) will reduce caibachol-induced vocalization. A total of sixty rats were implanted with stainless steel guide cannulae in the AH/POA area. After recovery, animals were locally pretreated with 1) GABA (l-40ng), 2) picrotoxin (1 .5^g) or bicuculhne (0.03ng), or 3) sahne; before injection with carbachol (1 .5^g). The resulting vocalization was measured and quantitated. The results indicate that pretreatment with GABA or GABA antagonists had no significant effect on vocalization. Local pretreatment with GABA did not potentiate the vocal response as measured by its duration, latraicy, and total number of calls. Similarly, pretreatment with picrotoxin or bicuculline had no effects on the same measures of vocalization. The results suggest tfiat chohnoceptive neurons involved in the production of alarm calls are not under direct GABAergic control.

Biotransformation of aromatic hydrocarbons and organic sulphides by fungi

Toluene is converted to benzyl alcohol by the fungi Mortierella isabellina and Helminthosporium species; in the latter case, the product is further metabolized. Toluene-a -d 1 , toluene-a,a-d2, and toluene-a,a,a-d 3 have been used with Mortierellaisabellina in a series of experiments to determine both primary and secondary deuterium kinetic isotope effects for the enzymic benzylic hydroxylation reaction. The values obtained, intermolecular primary kH/kD = intramolecular p rim a r y kH r kD = 1. 0 2 + O. 0 5, and sec 0 n dar y k H I kD = 1. 37 .:!. 0.05, suggest a mechanism for the reaction involving benzylic proton removal from a radical intermediate in a non-symmetrical transition state. 2H NMR (30.7 MHz) studies using ethylbenzene-l,1-d 2 , 3 -fluoroethylbenzene-l,1-d 2 , 4 -fluoroethylbenzene-l,1-d 2 , and toluene-dB as substrates with Mortierella isabellina suggest, based on the observable differences in rates of conversion between the substrates, that the hydroxylation of hydrocarbons at the benzylic position proceeds via a one electron abstraction from the aromatic ring, giving a radical cation. A series of 1,3-oxathiolanes (eight) were incubated with Mortierella isabellina , Helminthosporium , Rhizopus arrhizus , and Aspergillus niger . Sulphoxides were obtained from Mortierella isabellina and Rhizopus arrhizus using the substrates 2-phenyl-, 2-methyl-2-phenyl-, and 2-phenyl-2-tert. butyl-l,3-oxathiolane. The relative stereochemistry of 2-methyl-2-phenyl-l,3-oxathiolan-l-oxide was assigned based on lH decoupling, n.O.e, 1 and H NMR experiments. The lH NMR (200 MHz) of the methylene protons of 2-methyl-2-phenyl-l,3-oxathiolan-l-oxide was used as a diagnostic standard in assigning the relative stereochemistry of 2-phenyl-l,3-oxathiolan-l-oxide and 2-phenyl-2-tert. butyl-l,3-oxathiolan-l-oxide. The sulphoxides obtained were consistent with an oxidation occurring from the opposite side of the molecule to the phenyl substituent.

Evolutionary synthesis of stochastic gene network models using feature-based search spaces

A feature-based fitness function is applied in a genetic programming system to synthesize stochastic gene regulatory network models whose behaviour is defined by a time course of protein expression levels. Typically, when targeting time series data, the fitness function is based on a sum-of-errors involving the values of the fluctuating signal. While this approach is successful in many instances, its performance can deteriorate in the presence of noise. This thesis explores a fitness measure determined from a set of statistical features characterizing the time series' sequence of values, rather than the actual values themselves. Through a series of experiments involving symbolic regression with added noise and gene regulatory network models based on the stochastic 'if-calculus, it is shown to successfully target oscillating and non-oscillating signals. This practical and versatile fitness function offers an alternate approach, worthy of consideration for use in algorithms that evaluate noisy or stochastic behaviour.

Social neuroendocrinology of competition

The relationship between testosterone concentrations and aggressive behaviour in studies of people has produced very inconsistent findings. However, one consistent fmding that has emerged is that competitive and aggressive interactions potentiate testosterone release in both human and non-human species. It has been argued that socially-induced alterations in testosterone concentrations may function to influence ongoing and/or future social behaviour. Nonetheless, few studies have empirically tested this hypothesis. The current series of experiments was designed to address the extent to which competitioninduced fluctuations in testosterone concentrations were associated with ongoing and/or subsequent social behaviour. In Study 1, men (n = 38) provided saliva samples prior to, and at the conclusion of, the Point Subtraction Aggression Paradigm (PSAP). Although baseline testosterone concentrations were not related to aggressive behaviour, there was a positive correlation between change in testosterone and aggressive behaviour such that men who were most aggressive on the PSAP demonstrated the largest increase in testosterone concentrations. Furthermore, a rise in testosterone during the PSAP predicted willingness to choose a subsequent competitive task. In Study 2, men and women provided saliva samples prior to and after competing against a same-sex opponent on the Number Tracing Task (NTT). The outcome of the competition was rigged such that half of the individuals won most of the races, while the other half lost most of the races, thus experimentally creating a winner and loser in the laboratory. Following the competitive interaction, men and women played the PSAP with their same-sex partner. Results indicated that men selected the aggressive response (but not reward or protection responses), more frequently than women. For men assigned to the loss condition, an increase in testosterone concentrations in response to the NTT predicted subsequent aggressive behaviour. For men assigned to the win condition, an increase in testosterone concentrations in response to the NTT predicted subsequent aggressive behaviour, but only among those men who scored high on trait dominance. Change in testosterone and trait dominance did not predict aggressive behaviour in women. In Study 3, men provided saliva samples prior to, during, and at the end of the PSAP. They were randomly assigned to one of four experimental conditions that differed in the extent to which they were provoked and whether they received reward for behaving aggressively (i.e., stealing points). Results indicated that baseline testosterone concentrations did not correlate with aggression in any of the experimental conditions. Consistent with Study 1, there was a positive correlation between change in testosterone and aggressive behaviour among men who were provoked, but did not receive reward for aggression (i.e., reactive condition). Men who were provoked but did not receive reward for aggression enjoyed the task the most and were more likely to choose the competitive versus non-competitive task relative to men assigned to the other experimental conditions. Also, individual differences in aggressive behaviour among these men were positively correlated with the extent to which they enjoyed the task. Together, these studies indicate that testosterone dynamics within the context of competition influence subsequent competitive and aggressive behaviours in humans and that testosterone may be a marker of the intrinsically rewarding nature of costly aggressive behaviour.

The development of norm-based coding and category-specific face prototypes : an examination of 5- and 8-year-olds' face space

The present set of experiments was designed to investigate the organization and refmement of young children's face space. Past research has demonstrated that adults encode individual faces in reference to a distinct face prototype that represents the average of all faces ever encountered. The prototype is not a static abstracted norm but rather a malleable face average that is continuously updated by experience (Valentine, 1991); for example, following prolonged viewing of faces with compressed features (a technique referred to as adaptation), adults rate similarly distorted faces as more normal and more attractive (simple attractiveness aftereffects). Recent studies have shown that adults possess category-specific face prototypes (e.g., based on race, sex). After viewing faces from two categories (e.g., Caucasian/Chinese) that are distorted in opposite directions, adults' attractiveness ratings simultaneously shift in opposite directions (opposing aftereffects). The current series of studies used a child-friendly method to examine whether, like adults, 5- and 8-year-old children show evidence for category-contingent opposing aftereffects. Participants were shown a computerized storybook in which Caucasian and Chinese children's faces were distorted in opposite directions (expanded and compressed). Both before and after adaptation (i.e., reading the storybook), participants judged the normality/attractiveness of a small number of expanded, compressed, and undistorted Caucasian and Chinese faces. The method was first validated by testing adults (Experiment I ) and was then refined in order to test 8- (Experiment 2) and 5-yearold (Experiment 4a) children. Five-year-olds (our youngest age group) were also tested in a simple aftereffects paradigm (Experiment 3) and with male and female faces distorted in opposite directions (Experiment 4b). The current research is the first to demonstrate evidence for simple attractiveness aftereffects in children as young as 5, thereby indicating that similar to adults, 5-year-olds utilize norm-based coding. Furthermore, this research provides evidence for racecontingent opposing aftereffects in both 5- and 8-year-olds; however, the opposing aftereffects demonstrated by 5-year-olds were driven largely by simple aftereffects for Caucasian faces. The lack of simple aftereffects for Chinese faces in 5-year-olds may be reflective of young children's limited experience with other-race faces and suggests that children's face space undergoes a period of increasing differentiation over time with respect to race. Lastly, we found no evidence for sex -contingent opposing aftereffects in 5-year-olds, which suggests that young children do not rely on a fully adult-like face space even for highly salient face categories (i.e., male/female) with which they have comparable levels of experience.

The influence of emotional body posture on adults' and 8-year-olds' perception facial expressions

The present set of experiments was designed to investigate the development of children's sensitivity of facial expressions observed within emotional contexts. Past research investigating both adults' and children's perception of facial expressions has been limited primarily to the presentation of isolated faces. During daily social interactions, however, facial expressions are encountered within contexts conveying emotions (e.g., background scenes, body postures, gestures). Recently, research has shown that adults' perception of facial expressions is influenced by these contexts. When emotional faces are shown in incongruent contexts (e.g., when an angry face is presented in a context depicting fear) adults' accuracy decreases and their reaction times increase (e.g., Meeren et a1. 2005). To examine the influence of emotional body postures on children's perception of facial expressions, in each of the experiments in the current study adults and 8-year-old children made two-alternative forced choice decisions about facial expressions presented in congruent (e.g., a face displayed sadness on a body displaying sadness) and incongruent (e.g., a face displaying fear on a body displaying sadness) contexts. Consistent with previous studies, a congruency effect (better performance on congruent than incongruent trials) was found for both adults and 8-year-olds when the emotions displayed by the face and body were similar to each other (e.g., fear and sad, Experiment l a ) ; the influence of context was greater for 8-year-olds than adults for these similar expressions. To further investigate why the congruency effect was larger for children than adults in Experiment 1 a, Experiment 1 b was conducted to examine if increased task difficulty would increase the magnitude of adults' congruency effects. Adults were presented with subtle facial and despite successfully increasing task difficulty the magnitude of the. congruency effect did not increase suggesting that the difference between children's and adults' congruency effects in Experiment l a cannot be explained by 8-year-olds finding the task difficult. In contrast, congruency effects were not found when the expressions displayed by the face and body were dissimilar (e.g., sad and happy, see Experiment 2). The results of the current set of studies are examined with respect to the Dimensional theory and the Emotional Seed model and the developmental timeline of children's sensitivity to facial expressions. A secondary aim of the series of studies was to examine one possible mechanism underlying congruency effe cts-holistic processing. To examine the influence of holistic processing, participants completed both aligned trials and misaligned trials in which the faces were detached from the body (designed to disrupt holistic processing). Based on the principles of holistic face processing we predicted that participants would benefit from misalignment of the face and body stimuli on incongruent trials but not on congruent trials. Collectively, our results provide some evidence that both adults and children may process emotional faces and bodies holistically. Consistent with the pattern of results for congruency effects, the magnitude of the effect of misalignment varied with the similarity between emotions. Future research is required to further investigate whether or not facial expressions and emotions conveyed by the body are perceived holistically.