Localization and functional characterization of iridoid biosynthetic genes in Catharanthus roseus /

Catharanthus roseus is the sole biological source of the medicinal compounds vinblastine and vincristine. These chemotherapeutic compounds are produced in the aerial organs of the plant, however they accumulate in small amounts constituting only about 0.0002% of the fresh weight of the leaf. Their limited biological supply and high economical value makes its biosynthesis important to study. Vinblastine and vincristine are dimeric monoterpene indole alkaloids, which consists of two monomers vindoline and catharanthine. The monoterpene indole alkaloids (MIA's) contain a monoterpene moiety which is derived from the iridoid secologanin and an indole moiety tryptamine derived from the amino acid tryptophan. The biosynthesis of the monoterpene indole alkaloids has been localized to at least three cell types namely, the epidermis, the laticifer and the internal phloem assisted parenchyma. Carborundum abrasion (CA) technique was developed to selectively harvest epidermis enriched plant material. This technique can be used to harvest metabolites, protein or RNA. Sequencing of an expressed sequence tagged (EST) library from epidermis enriched mRNA demonstrated that this cell type is active in synthesizing a variety of secondary metabolites namely, flavonoids, lipids, triterpenes and monoterpene indole alkaloids. Virtually all of the known genes involved in monterpene indole alkaloid biosynthesis were sequenced from this library.This EST library is a source for many candidate genes involved in MIA biosynthesis. A contig derived from 12 EST's had high similarity (E'^') to a salicylic acid methyltransferase. Cloning and functional characterization of this gene revealed that it was the carboxyl methyltransferase imethyltransferase (LAMT). In planta characterization of LAMT revealed that it has a 10- fold enrichment in the leaf epidermis as compared to the whole leaf specific activity. Characterization of the recombinant enzyme revealed that vLAMT has a narrow substate specificity as it only accepts loganic acid (100%) and secologanic acid (10%) as substrates. rLAMT has a high Km value for its substrate loganic acid (14.76 mM) and shows strong product inhibition for loganin (Kj 215 |iM). The strong product inhibition and low affinity for its substrate may suggest why the iridoid moiety is the limiting factor in monoterpene indole alkaloid biosynthesis. Metabolite profiling of C. roseus organs shows that secologanin accumulates within these organs and constitutues 0.07- 0.45% of the fresh weight; however loganin does not accumulate within these organs suggesting that the product inhibition of loganin with LAMT is not physiologically relevant. The limiting factor to iridoid and MIA biosynthesis seems to be related to the spatial separation of secologanin and the MIA pathway, although secologanin is synthesized in the epidermis, only 2-5% of the total secologanin is found in the epidermis while the remaining secologanin is found within the leaf body inaccessable to alkaloid biosynthesis. These studies emphasize the biochemical specialization of the epidermis for the production of secondary metabolites. The epidermal cells synthesize metabolites that are sequestered within the plant and metabolites that are secreted to the leaf surface. The secreted metabolites comprise the epidermome, a layer separating the plant from its environment.

The Structural and Functional Changes of Blood Vessels during Aging

The vascular adventitia is recognized as a dynamic mediator of vascular structure and function, yet its role in aging is not understood. The purpose of this thesis was to examine the age-related changes of the vascular adventitia and determine the underlying mediators responsible. Male Sprague-Dawley rats were aged to 15, 30, 50 and 80 weeks before being anesthetised and euthanized by exsanguination. Thoracic aortas, mesenteric and pudental arteries were isolated, formalin fixed, and embedded in paraffin then sectioned at 5μm. Vessels were examined by microscopy and protein expression was determined by indirect immunofluorescence. The thickness of the adventitia increased dramatically with age. Immunofluorescence revealed a robust expression of endothelin system proteins in the adventitia. Additionally, extracellular matrix proteins collagen and fibronectin, and the proliferation marker Ki67 showed strong adventitial origin. The changes observed in the vascular adventitia with aging clearly demonstrate an important role in the process of vascular aging.

The structural and functional changes of blood vessels during aging

The vascular adventitia is recognized as a dynamic mediator of vascular structure and function, yet its role in aging is not understood. The purpose of this thesis was to examine the age-related changes of the vascular adventitia and determine the underlying mediators responsible. Male Sprague-Dawley rats were aged to 15,30,50 and 80 weeks before being anesthetised and euthanized by exsanguination. Thoracic aortas, mesenteric and pudental arteries were isolated, formalin fixed, and embedded in paraffin then sectioned at 51lm. Vessels were examined by microscopy and protein expression was determined by indirect immunofluorescence. The thickness of the adventitia increased dramatically with age. Immunofluorescence revealed a robust expression of endothelin system proteins in the adventitia. Additionally, extracellular matrix proteins collagen and fibronectin, and the proliferation marker Ki67 showed strong adventitial origin. The changes observed in the vascular adventitia with aging clearly demonstrate an important role in the process of vascular aging.

The Impact of Directed Mirror Focus and Technique Cues on Psychological, Cognitive and Emotional Exercise Correlates in an Introductory Weight Training Orientation

The purpose of the study was to investigate whether teaching inactive and low active women to use mirrors for form and technique purposes could lessen the negative impact of mirrors on self-presentational concerns, affect, and self-efficacy. Eligible women (N = 82) underwent a one-on-one weight training orientation with a personal trainer. Participants were randomized into one of four experimental groups, each unique in the type of feedback (general or technique-specific) and the degree of focus on the mirror for technique reinforcement. Questionnaires assessed study outcomes pre- and post-orientation. Results indicated groups did not significantly differ on any post-condition variables, when controlling for pre-condition values (all p’s >.05). All groups showed outcome improvements following the orientation. This suggests that during a complex task, a personal trainer who emphasizes form and technique can facilitate improvements to psychological outcomes in novice exercisers, independent of the presence of mirrors or directional cues provided.

Predictors of academic achievement and social competence in adolescence : facilitation, inhibition and general cognitive processing /

The relevance of attentional measures to cognitive and social adaptive behaviour was examined in an adolescent sample. Unlike previous research, the influence of both inhibitory and facilitory aspects of attention were studied. In addition, contributions made by these attentional processes were compared with traditional psychometric measures of cognitive functioning. Data were gathered from 36 grade 10 and 1 1 high school students (20 male and 16 female students) with a variety of learning and attentional difficulties. Data collection was conducted in the course of two testing sessions. In the first session, students completed questionnaires regarding their medical history, and everyday behaviours (the Brock Adaptive Functioning Questionnaire), along with non-verbal problem solving tasks and motor speed tasks. In the second session, students performed working memory measures and computer-administered tasks assessing inhibitory and facilitory aspects of attention. Grades and teacher-rated measures of cognitive and social impulsivity were also gathered. Results indicate that attentional control has both cognitive and social/emotional implications. Performance on negative priming and facilitation trials from the Flanker task predicted grades in core courses, social functioning measures, and cognitive and social impulsivity ratings. However, beneficial effects for academic and social functioning associated with inhibition were less prevalent in those demonstrating a greater ability to respond to facilitory cues. There was also some evidence that high levels of facilitation were less beneficial to academic performance, and female students were more likely to exceed optimal levels of facilitory processing. Furthermore, lower negative priming was ''S'K 'i\':y-: -'*' - r " j«v ; ''*.' iij^y Inhibition, Facilitation and Social Competence 3 associated with classroom-rated distraction and hyperactivity, but the relationship between inhibition and social aspects of impulsivity was stronger for adolescents with learning or reading problems, and the relationship between inhibition and cognitive impulsivity was stronger for male students. In most cases, attentional measures were predictive of performance outcomes independent of traditional psychometric measures of cognitive functioning. >,, These findings provide support for neuropsychological models linking inhibition to control of interference and arousal, and emphasize the fundamental role of attention in everyday adolescent activities. The findings also warrant further investigation into the ways which inhibitory and facilitory attentional processes interact, and the contextdependent nature of attentional control.associated with classroom-rated distraction and hyperactivity, but the relationship between inhibition and social aspects of impulsivity was stronger for adolescents with learning or reading problems, and the relationship between inhibition and cognitive impulsivity was stronger for male students. In most cases, attentional measures were predictive of performance outcomes independent of traditional psychometric measures of cognitive functioning. >,, These findings provide support for neuropsychological models linking inhibition to control of interference and arousal, and emphasize the fundamental role of attention in everyday adolescent activities. The findings also warrant further investigation into the ways which inhibitory and facilitory attentional processes interact, and the contextdependent nature of attentional control.

Individual differences in risk-taking and associated characteristics : a test of specificity in executive functions /

Multiple measures have been devised by clinicians and theorists from many different backgrounds for the purpose of assessing the influence of the frontal lobes on behaviour. Some utilize self-report measures to investigate behavioural characteristics such as risktaking, sensation seeking, impulsivity, and sensitivity to reward and punishment in an attempt to understand complex human decision making. Others rely more on neuroimaging and electrophysiological investigation involving experimental tasks thought to demonstrate executive functions in action, while other researchers prefer to study clinical populations with selective damage. Neuropsychological models of frontal lobe functioning have led to a greater appreciation of the dissociations among various aspects of prefrontal cortex function. This thesis involves (1) an examination of various psychometric and experimental indices of executive functions for coherence as one would predict on the basis of highly developed neurophysiological models of prefrontal function, particularly those aspects of executive function that involve predominantly cognitive abilities versus processes characterized by affect regulation; and (2) investigation of the relations between risk-taking, attentional abilties and their associated characteristics using a neurophysiological model of prefrontal functions addressed in (1). Late adolescence is a stage in which the prefrontal cortices undergo intensive structural and functional maturational changes; this period also involves increases in levels of risky and sensation driven behaviours, as well as a hypersensitivity to reward and a reduction in inhibition. Consequently, late adolescence spears to represent an ideal developmental period in which to examine these decision-making behaviours due to the maximum variability of behavioural characteristics of interest. Participants were 45 male undergraduate 18- to 19-year olds, who completed a battery of measures that included self-report, experimental and behavioural measures designed to assess particular aspects of prefrontal and executive functioning. As predicted, factor analysis supported the grouping of executive process by type (either primarily cognitive or affective), conforming to the orbitofrontal versus dorsolateral typology; risk-taking and associated characteristics were associated more with the orbitofrontal than the dorsolateral factor, whereas attentional and planning abilities tended to correlate more strongly with the dorsolateral factor. Results are discussed in light of future assessment, investigation and understanding of complex human decision-making and executive functions. Implications, applications and suggestions for future research are also proposed.

Investigating the effects of arousal state on cognitive performance in individuals with and without mild head injury

We examined the cognitive and emotional sequelae following mild head injury (MHI; e.g., concussion) in high-functioning individuals and whether persons with MHI pre~ent, both physiologically and via self-report, in a manner different from (i.e., underaroused) that of persons who have no history of head injury. We also investigated the effect arousal state ~as on the cognitive performance of this population. Using a quasiexperimental research design (N = 91), we examined changes in attention, working memory, and cognitive flexibility (subtests ofthe WAIS-III, 1997,WMS-III, 1997, & DKEFS, 2002) as a function of manipulated arousal (i.e., induced psychosocial stress/activation; reduced activation/relaxation). In addition to self-reported arousal and state anxiety (State-Trait Anxiety Inventory; Speilberger, 1983a) measures, physiological indices of arousal state (i.e., electrodermal responsivity, heart rate, and respiration activity) were recorded (via Polygraph Professional Suite, 2008) across a 2.5 hour interval while completing various cognitive tasks. Students also completed the Post-concussive Symptom Checklist (Gouvier et aI., 1992). The results demonstrate that university students who report a history ofMHI (i.e., "altered state of consciousness") experience significantly lower levels of anxiety, were physiologically underaroused, and were less responsive to stressors in their environment, compared to their non-~HI cohorts. As expected, cognitive flexibility (but not other neuropsychological measures of cognition) was advantaged with increased stress, and disadvantaged with reduced stress, in persons with reported MHI, but not for those without reported MHI which provided limited support for our hypothesis. Further, university students who had no complaints related to their previous MHI endorsed a greater number of traditional post-concussive symptoms in terms of intensity, duration and frequency as compared to students who did not report a MHI. The underarousal in traumatic brain injury has been associated with (ventromedial prefrontal cortex) VMPFC disruption and may be implicated in MHI generally. Students who report sustaining a previous MHI may be less able to physiologically respond and/or cognitively appraise, stressful experiences as compared to their no-MHI cohort and experience persistent, long-lasting consequences despite the subtle nature of a history of head injury.

A comparison of functional behaviour assessment rating scales in a sample of children and youth with autism spectrum disorders

This study sought to compare the results of the Motivation Assessment Scale (MAS; Durand & Crimmins, 1988), Questions About Behavior Function Scale (QABF; Matson & Vollmer, 1996) and Functional Analysis Screening Tool (FAST; Iwata & Deleon, 1996), when completed by parent informants in a sample of children and youth with autism spectrum disorders (ASD) who display challenging behaviour. Results indicated that there was low agreement between the functional hypotheses derived from each of three measures. In addition, correlations between functionally analogous scales were substantially lower than expected, while correlations between non-analogous subscales were stronger than anticipated. As indicated by this study, clinicians choosing to use FBA questionnaires to assess behavioural function, may not obtain accurate functional hypotheses, potentially resulting in ineffective intervention plans. The current study underscores the caution that must be taken when asking parents to complete these questionnaires to determine the function(s) of challenging behaviour for children/youth with ASD.

Functional analysis and treatment of OCD-related behaviour in a child with Autism Spectrum Disorder

Research indicates that Obsessive-Compulsive Disorder (OCD; DSM-IV-TR, American Psychiatric Association, 2000) is the second most frequent disorder to coincide with Autism Spectrum Disorder (ASD; Leyfer et aI., 2006). Excessive collecting and hoarding are also frequently reported in children with ASD (Berjerot, 2007). Although functional analysis (Iwata, Dorsey, Slifer, Bauman, & Richman, 1982/1994) has successfully identified maintaining variables for repetitive behaviours such as of bizarre vocalizations (e.g., Wilder, Masuda, O'Connor, & Baham, 2001), tics (e.g., Scotti, Schulman, & Hojnacki, 1994), and habit disorders (e.g., Woods & Miltenberger, 1996), extant literature ofOCD and functional analysis methodology is scarce (May et aI., 2008). The current studies utilized functional analysis methodology to identify the types of operant functions associated with the OCD-related hoarding behaviour of a child with ASD and examined the efficacy of function-based intervention. Results supported hypotheses of automatic and socially mediated positive reinforcement. A corresponding function-based treatment plan incorporated antecedent strategies and differential reinforcement (Deitz, 1977; Lindberg, Iwata, Kahng, and DeLeon, 1999; Reynolds, 1961). Reductions in problem behaviour were evidenced through use of a multiple baseline across behaviours design and maintained during two-month follow-up. Decreases in symptom severity were also discerned through subjective measures of treatment effectiveness.

Disambiguating the Effects of Social Instability Stress in Adolescence on Learning and Memory Tasks that Involve the Medial Prefrontal Cortex and Hippocampus

Exposure to chronic stress can alter the structure and function of brain regions involved in learning and memory, and these effects are typically long-lasting if the stress occurs during sensitive periods of development. Until recently, adolescence has received relatively little attention as a sensitive period of development, despite marked changes in behaviour, heightened reactivity to stressors, and cognitive and neural maturation. Therefore, the purpose of the present study was to investigate the long-term effects of chronic stress in adolescence on two spatial learning and memory tasks (Morris water maze and Spatial Object Location test) and on a working memory task (Delayed Alternation task). Male rats were randomly assigned to chronic social instability stress (SS; daily 1 hour isolation and subsequent change of cage partner between postnatal days 30 and 45) or to a no-stress control group (CTL). During acquisition learning in the Morris water maze task, SS rats demonstrated impaired long-term memory for the location of the hidden escape platform compared to CTL rats, although the impairment was only seen after the first day of training. Similarly, SS rats had impaired long-term memory in the Spatial Object Location test after a long delay (240 minutes), but not after shorter delays (15 or 60 minutes) compared to CTL rats. On the Delayed Alternation task, which assessed working memory across delays ranging from 5 to 90 seconds, no group differences were observed. These results are partially in line with previous research that revealed adult impairment on spatial learning and memory tasks after exposure to chronic social instability stress in adolescence. The observed deficits, however, appear to be limited to long-term memory as no group differences were observed during brief periods of retention.

Autonomic and Electrophysiological Correlates of Cognitive Control in Aging

This thesis tested a model of neurovisceral integration (Thayer & Lane, 2001) wherein parasympathetic autonomic regulation is considered to play a central role in cognitive control. We asked whether respiratory sinus arrhythmia (RSA), a parasympathetic index, and cardiac workload (rate pressure product, RPP) would influence cognition and whether this would change with age. Cognitive control was measured behaviourally and electrophysiologically through the error-related negativity (ERN) and error positivity (Pe). The ERN and Pe are thought to be generated by the anterior cingulate cortex (ACC), a region involved in regulating cognitive and autonomic control and susceptible to age-related change. In Study 1, older and younger adults completed a working memory Go/NoGo task. Although RSA did not relate to performance, higher pre-task RPP was associated with poorer NoGo performance among older adults. Relations between ERN/Pe and accuracy were indirect and more evident in younger adults. Thus, Study 1 supported the link between cognition and autonomic activity, specifically, cardiac workload in older adults. In Study 2, we included younger adults and manipulated a Stroop task to clarify conditions under which associations between RSA and performance will likely emerge. We varied task parameters to allow for proactive versus reactive strategies, and motivation was increased via financial incentive. Pre-task RSA predicted accuracy when response contingencies required maintenance of a specific item in memory. Thus, RSA was most relevant when performance required proactive control, a metabolically costly strategy that would presumably be more reliant on autonomic flexibility. In Study 3, we included older adults and examined RSA and proactive control in an additive factors framework. We maintained the incentive and measured fitness. Higher pre-task RSA among older adults was associated with greater accuracy when proactive control was needed most. Conversely, performance of young women was consistently associated with fitness. Relations between ERN/Pe and accuracy were modest; however, isolating ACC activity via independent component analysis allowed for more associations with accuracy to emerge in younger adults. Thus, performance in both groups appeared to be differentially dependent on RSA and ACC activation. Altogether, these data are consistent with a neurovisceral integration model in the context of cognitive control.