<p>Enzyme induction is a process whereby a molecule enhances the expression of enzymes. If the affected enzymes are involved in the elimination of a drug, this may result in a drug interaction. Induction is therefore of major concern during drug development and in clinical practice. </p><p>The induction process depends on the half-life of the induced enzyme, the pharmacokinetics of the inducing agent, and the relationship between the inducer’s concentration and the induction stimulus. The aim of the conducted research was to investigate these key aspects of enzyme induction and the consequences that induction has for substrate elimination.</p><p>Successful investigations of the induction process presuppose the existence of appropriate methods for the estimation of the metabolic activity. Enzyme activity measurements can be conducted in tissues with low enzyme content using the analytical method presented here. </p><p>A model was developed describing the changes in the pharmacokinetics of clomethiazole and its metabolite NLA-715, that are attributable to carbamazepine induction. The consequences of the induction was explained using a mechanistic approach, acknowledging food-induced changes in the blood flow to the liver, and interpreting in vitro generated metabolic information.</p><p>The time course of the induction process was examined in two investigations. In the first of these, the pharmacokinetics of the autoinducing drug phenobarbital and its effect on several enzymes were described in rats. This was accomplished by integrating the bidirectional interaction between drug and enzymes in a mechanistic manner. In the final investigation, the time course of the increase and cessation in enzyme activity was studied in healthy volunteers treated with carbamazepine. This investigation allowed the half-lives of CYP3A and CYP1A2 to be estimated. </p><p>The key aspects of the enzyme induction process have been examined using mechanistic induction models. These novel models improve the understanding of the induction process and its consequences for substrate elimination.</p>

Enzyme induction is a process whereby a molecule enhances the expression of enzymes. If the affected enzymes are involved in the elimination of a drug, this may result in a drug interaction. Induction is therefore of major concern during drug development and in clinical practice. The induction process depends on the half-life of the induced enzyme, the pharmacokinetics of the inducing agent, and the relationship between the inducer’s concentration and the induction stimulus. The aim of the conducted research was to investigate these key aspects of enzyme induction and the consequences that induction has for substrate elimination. Successful investigations of the induction process presuppose the existence of appropriate methods for the estimation of the metabolic activity. Enzyme activity measurements can be conducted in tissues with low enzyme content using the analytical method presented here. A model was developed describing the changes in the pharmacokinetics of clomethiazole and its metabolite NLA-715, that are attributable to carbamazepine induction. The consequences of the induction was explained using a mechanistic approach, acknowledging food-induced changes in the blood flow to the liver, and interpreting in vitro generated metabolic information. The time course of the induction process was examined in two investigations. In the first of these, the pharmacokinetics of the autoinducing drug phenobarbital and its effect on several enzymes were described in rats. This was accomplished by integrating the bidirectional interaction between drug and enzymes in a mechanistic manner. In the final investigation, the time course of the increase and cessation in enzyme activity was studied in healthy volunteers treated with carbamazepine. This investigation allowed the half-lives of CYP3A and CYP1A2 to be estimated. The key aspects of the enzyme induction process have been examined using mechanistic induction models. These novel models improve the understanding of the induction process and its consequences for substrate elimination.

Analysis of cuticular wax from Kalanchoe daigremontiana leaves was performed to identify the constituent components within the wax, determine how these changed during leaf ontogenesis, and discover how they were distributed within the cuticle. Analysis of extracted cuticular wax by gas chromatography, mass spectrometry, and comparison with authentic standards led to the identification of triterpenoids including glutinol, friedelin, germanicol, epifriedelanol, glutinol acetate and β-amyrin as well as very long chain fatty acid (VLCFA) derivatives including alkanes, primary alcohols, aldehydes, fatty acids, and alkyl esters. Cuticular wax composition in young K. daigremontiana leaves was dominated by triterpenoids, which made up over 70% of the lipid soluble compounds. During leaf ontogenesis, wax composition changed to include a higher proportion of VLCFA derivatives, which made up approximately 50% of cuticular wax in mature leaves. The most abundant triterpenoids in the wax were glutinol and friedelin, both fairly uncommon pentacyclic triterpenoids with a complex proposed biosynthetic mechanism. Tritriacontane (C33 alkane) was the most abundant compound within the VLCFA derivatives. Cuticular wax accumulation was found to correspond well to leaf growth, with both processes slowing at the same time. Variations in the ratio of friedelin-like compounds to glutinol-like compounds during leaf ontogenesis suggest the presence more than one active triterpenoid synthase enzyme in the leaves of K. daigremontiana. VLCFA compounds were found mainly in the epicuticular wax on both the adaxial and abaxial surfaces, while triterpenoids were relatively more abundant in the intracuticular layer. Two different epicuticular wax crystal forms were observed by scanning electron microscopy (SEM) which can be described as platelets with sinuate margins and twisted ribbons. Based on SEM and chemical data as well as previous reports of crystal composition, it is hypothesized that each crystal type has a unique composition, with the platelets containing one or more triterpenoids and the twisted ribbons containing alkanes and other VLCFA derivatives. Confirmation of this hypothesis will have to await further investigation. This research provides information that will aid in the larger goals of characterizing a glutinol or friedelin synthase and understanding the gradients established within epicuticular and intracuticular wax layers.

Analysis of cuticular wax from Kalanchoe daigremontiana leaves was performed to identify the constituent components within the wax, determine how these changed during leaf ontogenesis, and discover how they were distributed within the cuticle. Analysis of extracted cuticular wax by gas chromatography, mass spectrometry, and comparison with authentic standards led to the identification of triterpenoids including glutinol, friedelin, germanicol, epifriedelanol, glutinol acetate and β-amyrin as well as very long chain fatty acid (VLCFA) derivatives including alkanes, primary alcohols, aldehydes, fatty acids, and alkyl esters. Cuticular wax composition in young K. daigremontiana leaves was dominated by triterpenoids, which made up over 70% of the lipid soluble compounds. During leaf ontogenesis, wax composition changed to include a higher proportion of VLCFA derivatives, which made up approximately 50% of cuticular wax in mature leaves. The most abundant triterpenoids in the wax were glutinol and friedelin, both fairly uncommon pentacyclic triterpenoids with a complex proposed biosynthetic mechanism. Tritriacontane (C33 alkane) was the most abundant compound within the VLCFA derivatives. Cuticular wax accumulation was found to correspond well to leaf growth, with both processes slowing at the same time. Variations in the ratio of friedelin-like compounds to glutinol-like compounds during leaf ontogenesis suggest the presence more than one active triterpenoid synthase enzyme in the leaves of K. daigremontiana. VLCFA compounds were found mainly in the epicuticular wax on both the adaxial and abaxial surfaces, while triterpenoids were relatively more abundant in the intracuticular layer. Two different epicuticular wax crystal forms were observed by scanning electron microscopy (SEM) which can be described as platelets with sinuate margins and twisted ribbons. Based on SEM and chemical data as well as previous reports of crystal composition, it is hypothesized that each crystal type has a unique composition, with the platelets containing one or more triterpenoids and the twisted ribbons containing alkanes and other VLCFA derivatives. Confirmation of this hypothesis will have to await further investigation. This research provides information that will aid in the larger goals of characterizing a glutinol or friedelin synthase and understanding the gradients established within epicuticular and intracuticular wax layers.

An entire field of research was born when a paper entitled ‘On the education of muscular control and power’ first discussed a “psychical rather than a physical” bilateral adaptation to a unilateral training program. Although the true relevance of this paper would not be recognized for over a century, its novel findings, describing adaptations resulting from physical and skilled training, continue to influence scientific literature to this day. Most notably, Scripture coined the term ‘cross-education’ to describe the improvement in strength or functional performance of not only the trained limb but also in the untrained contralateral limb. Recently, unilateral training or ‘cross-education’ has been highlighted as a possible rehabilitation strategy during recovery from unilateral injuries. However, a number of limitations must be addressed within the scientific literature in order to properly apply unilateral resistance training as an effective rehabilitation strategy. Therefore, the primary goal of this dissertation was to address a number of fundamental issues related to optimizing unilateral resistance training. One such issue is knowledge on the time course of strength increase during unilateral resistance training. The primary purpose of Chapter 2 was to characterize the time-course of strength changes in both the trained and untrained limbs during unilateral handgrip training. Experiment 1 assessed the time-course with a ‘traditional’ training protocol (3x/week for 6 weeks: 18 total sessions) while Experiment 2 assessed a “compressed” protocol in which the number of sessions and contractions were matched but participants trained for eighteen consecutive days. An anticipated outcome was the determination of the minimum number of sessions required to induce contralateral strength gains in the upper limb. A secondary purpose of this study was to examine whether spinally-mediated adaptations in muscle afferent reflex pathways occur after unilateral handgrip training. Experiment 1 indicated six weeks of handgrip training significantly increased force output in both trained and untrained limbs. This strength increase was accompanied by changes in the maximal muscle activation in the trained limb only. Time course data indicated the trained limb was significantly stronger than baseline after the 3rd week of training (session 9) while the untrained limb was stronger after 5 weeks (15 sessions) of unilateral handgrip training. Interestingly, the rate at which strength increased in the untrained limb was similar to the trained side. These strength increases were also accompanied by significant changes in the current needed to produce H@50 in the trained, and Hmax in both the trained and untrained limb indicating alterations in spinal cord excitability. Experiment 2 showed a similar number of sessions was needed to induce significant strength gains in the untrained limb. This indicates training without rest days may be the most efficient protocol within a clinical population when the trained limb is not the focus of recovery. It remains necessary to determine if specific strategies can be employed to optimize unilateral resistance training interventions to increase strength gains. To date, no study has directly assessed the relative contribution of afferent pathways to cross-education. Cutaneous feedback from the skin provides perceptual information about joint position and movement. Unilateral training involves forceful contractions that activate cutaneous receptors in the skin, producing widespread and powerful effects between limbs. Providing “enhanced” cutaneous stimulation during unilateral contractions may alter excitability of interlimb reflex pathways, modifying the contralateral increase in strength. Therefore, the purpose of Chapter 3 was to determine the relative contribution of cutaneous afferent pathways as a mechanism of cross-education by directly assessing if unilateral cutaneous stimulation alters ipsilateral and contralateral strength gains. Participants were randomly assigned to either a voluntary contraction (TRAIN), cutaneous stimulation (STIM), or cutaneous stimulation during voluntary contraction (TRAIN+STIM) group. Each participant completed 6 sets of 8 reps 3x/week for 5 weeks. TRAIN included unilateral maximal voluntary isometric contractions (MVCs) of the wrist extensors. STIM training included cutaneous stimulation (2xRT for 3sec @ 50Hz) of the superficial radial (SR) nerve at the wrist only. TRAIN+STIM included MVCs of the wrist extensors with SR stimulation provided for the duration of the contraction. Two pre-training and 1 post-training session assessed the relative increase in force output during MVCs for wrist flexion, wrist extension and handgrip strength. Results indicated unilateral wrist extension training alone (TRAIN) increased force output in both trained and untrained wrist extensors. Providing ‘enhanced’ sensory feedback via electrical stimulation during training (TRAIN+STIM) led to similar increases in strength in the trained limb compared to TRAIN. However, the major finding revealed that ‘enhanced’ feedback in the TRAIN+STIM group completely blocked interlimb strength transfer to the untrained wrist extensors. It appears the large mismatched sensory volley which was provided may have interfered with the integration of the appropriate sensory cues to the untrained cortex and impaired the ability to induce “cross-education”. It may be possible to enhance effects of training by altering excitability via apparel such as compression garments. Currently, it is unknown whether tactile input to the skin induced via compression apparel may alter transmission of muscle afferent feedback within a limb. Thus, the purpose of Chapter 4 was to examine if sustained input to the skin via compression garment modulates sensory feedback transmission in the upper limb using the Hoffmann (H-) reflex as a probe. The purpose of these experiments was to: 1) explore the effects of compression gear on sensory feedback transmission in the upper limb during a static task, and 2) if the task (locomotor vs. reaching) or phase of a movement differentially modulated this transmission of sensory information. Furthermore, differences in performance of the discrete reaching task were assessed to provide data on whether a compression garment leads to alteration in motor task performance. Combined results from both parts of the study suggest that tactile input provided to the skin via compression garments modulates the excitability of afferent connections independent of descending input. The alteration in excitability occurs across multiple sensory pathways and across multiple movement tasks. Interestingly, there was a significant reduction in the number of errors made during the reaching task, which provides preliminary evidence of an improved performance while wearing a compression garment. Therefore, the compression sleeve appears to increase precision and sensitivity at the joint where it is applied. Overall, these results address many fundamental questions which have previously limited effective translation for rehabilitative interventions. These results provide preliminary guidelines for subsequent strength training interventions to prescribe the optimal ‘dose’ of unilateral strength training to maximize benefits while minimizing intervention burden. These studies also help refine a unifying model of unilateral strength training to include contributions from central motor output as well as afferent feedback. These studies highlight the importance of appropriate sensory feedback during maximal force production and the impact that sensory information from the skin can have on motor output in the nervous system. / Graduate / 0317 / 0719 / 0382

In studies of enzyme kinetics, reaction time courses are often condensed into a single set of initial rates describing the rate at the start of the reaction. This set is then analysed with the Henri-Michaelis-Menten equation. However, this process necessarily removes information from experimental data and diminishes its statistical significance due to a reduction of available data points. Further, if the examined system does not approach steady-state rapidly, the application of the steady-state-assumption can lead to flawed conclusions. Here, the analysis of two complex enzyme systems by numerical integration of kinetic rate equations is demonstrated. DNA polymerase catalyses the synthesis of DNA in a reaction that involves two substrates, DNA template and dNTP, both of which are highly heterogeneous in nature. The tight binding of DNA to DNA polymerase and its polymer properties prohibit the application of the initial-rate approach. By combining an explicit DNA binding step with a steady-state dNTP incorporation on a template of finite length, the DNA binding parameters and the dNTP incorporation steady-state parameters were estimated from processive polymerisation data in a global regression analysis. This approach is described in Chapter 2 and the results are in good agreement with previously published values. Further properties were investigated in studies of the temperature dependence and solvent isotope dependence of the kinetics. The processive polymerisation of DNA template was monitored using the fluorophore PicoGreen in a simple and inexpensive method described in Chapter 3. The catalytic cycle of ethanolamine ammonia lyase involves the homoloysis of the Co-C bond within the intrinsic B12 cofactor. This homolysis results in the formation of a Co(II)-adenosyl radical intermediate, which can be monitored using stopped-flow spectroscopy. The stopped-flow transients observed for EAL and related enzymes have long been difficult to analyse and interpret, possibly due to rapid methyl group rotation on the substrate. In Chapter 4 of this thesis we were able to rationalise this behaviour using numerical integration of the rate equations of a branched 16-state-kinetic model to fit stopped-flow transients in a global regression analysis. We were able to determine some intrinsic rate constants, and showed that the initial hydrogen atom transfer step is unlikely to have an inflated primary kinetic isotope effect, despite previous claims. More generally, this study demonstrates that the numerical integration analysis used here is likely to be applicable to a broad range of enzyme reaction kinetics.

Analysis of cuticular wax from Kalanchoe daigremontiana leaves was performed to identify the constituent components within the wax, determine how these changed during leaf ontogenesis, and discover how they were distributed within the cuticle. Analysis of extracted cuticular wax by gas chromatography, mass spectrometry, and comparison with authentic standards led to the identification of triterpenoids including glutinol, friedelin, germanicol, epifriedelanol, glutinol acetate and β-amyrin as well as very long chain fatty acid (VLCFA) derivatives including alkanes, primary alcohols, aldehydes, fatty acids, and alkyl esters. Cuticular wax composition in young K. daigremontiana leaves was dominated by triterpenoids, which made up over 70% of the lipid soluble compounds. During leaf ontogenesis, wax composition changed to include a higher proportion of VLCFA derivatives, which made up approximately 50% of cuticular wax in mature leaves. The most abundant triterpenoids in the wax were glutinol and friedelin, both fairly uncommon pentacyclic triterpenoids with a complex proposed biosynthetic mechanism. Tritriacontane (C33 alkane) was the most abundant compound within the VLCFA derivatives. Cuticular wax accumulation was found to correspond well to leaf growth, with both processes slowing at the same time. Variations in the ratio of friedelin-like compounds to glutinol-like compounds during leaf ontogenesis suggest the presence more than one active triterpenoid synthase enzyme in the leaves of K. daigremontiana. VLCFA compounds were found mainly in the epicuticular wax on both the adaxial and abaxial surfaces, while triterpenoids were relatively more abundant in the intracuticular layer. Two different epicuticular wax crystal forms were observed by scanning electron microscopy (SEM) which can be described as platelets with sinuate margins and twisted ribbons. Based on SEM and chemical data as well as previous reports of crystal composition, it is hypothesized that each crystal type has a unique composition, with the platelets containing one or more triterpenoids and the twisted ribbons containing alkanes and other VLCFA derivatives. Confirmation of this hypothesis will have to await further investigation. This research provides information that will aid in the larger goals of characterizing a glutinol or friedelin synthase and understanding the gradients established within epicuticular and intracuticular wax layers. / Science, Faculty of / Chemistry, Department of / Graduate

The process of movement execution can be separated into two sections; the foreperiod and the response time. The foreperiod represents the time between the warning signal (WS) and the presentation of the imperative “go” signal, and the response time incorporates both the reaction time (RT) and the movement time (Schmidt & Lee, 2011). Transcranial magnetic stimulation (TMS) was used to probe corticospinal excitability (CE) which has been measured in a variety of RT tasks during both the foreperiod and the response time periods. The purpose of the two studies in this thesis was to measure when and at what rate changes in CE occur in both simple and complex tasks. The results of the first experiment indicated that CE levels quickly increased from baseline with the presentation of the WS. This was followed by a holding period in which CE was held constant until a decline in CE occurred prior to the presentation of the IS. This decline was followed by a rapid increase in CE as the movement was initiated and released. Importantly, even though levels of CE were decreasing relative to the start of the decline, participants were still in a heightened state as they prepared to release their movements. Furthermore, it is suggested that selective inhibitory control mechanisms were at least partly responsible for the decline prior to the IS. The results of the second experiment indicated that MEP amplitudes in a simple task were significantly larger compared to those in a complex task relative to both the IS and the onset of electromyography. These findings suggest that simple and complex tasks achieve differing levels of corticospinal excitability, and it is suggested that the complex requires the use of the cerebellum, which suppresses excitatory projections to the thalamus, and consequently to the motor cortex.

No description available.

Phenotypic plasticity is the ability of the genotype to express alternative phenotypes in response to different environmental conditions and this is considered to be an adaptation in which a species can survive and persist in a rapidly changing environment. Some grasshoppers and locusts are capable of expressing an extreme form of density-dependent phenotypic plasticity, known as locust phase polyphenism. At low population density, the individuals typically have a cryptic coloration as nymphs, are less active, and only seek out conspecifics for reproductive purposes. At high density, however, they develop a drastically different phenotype in which they have a conspicuous coloration, are much more active, and tend to stay together in large groups. The American Birdwing grasshopper, Schistocerca americana, is a non-swarming species related to the desert locust, S. gregaria, which shows density-dependent phenotypic plasticity in behavior, color, and morphology. In this thesis, I have identified the duration of crowding necessary for a 6th instar S. americana reared in the isolated condition to express the typical crowded behavior. The behavior changed after just one hour of crowding and the effect of crowding diminished after 48 hours to near-complete isolated behavior. In reverse, the crowded condition was isolated, but behavior did not significantly change over time. Gene expression of the following three genes suspected of having a role in behavior change were investigated based on studies of S. gregaria: protein kinase A (PKA), L-Tryptophan-5-monooxygenase (T-5), and Aromatic L-amino acid decarboxylase (Decarb). T-5 was up-regulated in the long-term isolated condition compared to the long-term crowded condition. T-5 and Decarb were up-regulated in isolated individuals that were crowded for 10 hours compared to the long-term isolated condition. This study represents a novel contribution in the study of phenotypic plasticity as it establishes the time course of behavioral and molecular plasticity in a non-swarming grasshopper for the first time.