971 resultados para Effective range
Resumo:
Though stadium style seating in large lecture theatres may suggest otherwise, effective teaching and learning is a not a spectator sport. A challenge in creating effective learning environments in both physical and virtual spaces is to provide optimal opportunity for student engagement in active learning. Queensland University of Technology (QUT) has developed the Open Web Lecture (OWL), a new web-based student response application, which seamlessly integrates a virtual learning environment within the physical learning space. The result is a blended learning experience; a fluid collaboration between academic and students connected to OWL via the University’s Wi-Fi using their own laptop or mobile web device. QUT is currently piloting the OWL application to encourage student engagement. OWL offers opportunities for participants to: • Post comments and questions • Reply to comments • "Like" comments • Poll students and review data • Review archived sessions. Many of these features instinctively appeal to student users of social networking media, yet avail the academic of control within the University network. Student privacy is respected through a system of preserving peer-peer anonymity, a functionality that seeks to address a traditional reluctance to speak up in large classes. The pilot is establishing OWL as an opportunity for engaging students in active learning opportunities by enabling • virtual learning in physical spaces for large group lectures, seminar groups, workshops and conferences • live collaborative technology connecting students and the academic via the wireless network using their own laptop or mobile device • an non- intimidating environment in which to ask questions • promotion of a sense of community • instant feedback • problem based learning. The student and academic response to OWL has been overwhelmingly positive, crediting OWL as an easy to use application, which creates effective learning opportunities though interactivity and immediate feedback. This poster and accompanying online presentation of the technology will demonstrate how OWL offers new possibilities for active learning in physical spaces by: • providing increased opportunity for student engagement • supporting a range of learners and learning activities • fostering blended learning experiences. The presentation will feature visual displays of the technology, its various interfaces and feedback including clips from interviews with students and academics participating in the early stages of the pilot.
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Purpose — The purpose of this chapter is to outline the findings of research into knowledge transfer across countries and culture and the implications of the findings for human resource practitioners. Methodology/approach — Use of a qualitative case study methodology approach was employed. Findings — The findings highlight the inherent difficulty of knowledge transfer and the broad range of factors that influence the knowledge transfer process and which are connected together in a complex and non-linear manner. Research implications — The research revealed insight into those managers most likely to be effective at knowledge transfer and what characteristics and experiences had supported this openness to knowledge from outside of their cultural context. The lessons from the research can be incorporated into the selection and development processes for expatriates. Originality value — The study affirmed the critical importance of face-to-face interaction in knowledge transfer. The research also resulted in the development of a practical model and tool, which pulls together the broad range of factors that impact on knowledge transfer.
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For the analysis of material nonlinearity, an effective shear modulus approach based on the strain control method is proposed in this paper by using point collocation method. Hencky’s total deformation theory is used to evaluate the effective shear modulus, Young’s modulus and Poisson’s ratio, which are treated as spatial field variables. These effective properties are obtained by the strain controlled projection method in an iterative manner. To evaluate the second order derivatives of shape function at the field point, the radial basis function (RBF) in the local support domain is used. Several numerical examples are presented to demonstrate the efficiency and accuracy of the proposed method and comparisons have been made with analytical solutions and the finite element method (ABAQUS).
Resumo:
Objective. The Effective Consumer Scale (EC-17) comprises 17 items measuring the main skills and behaviors people need to effectively manage their healthcare. We tested the responsiveness of the EC-17. Methods. Participants, in 2 waves of a 6-week Arthritis Self-Management Program (ASMP) from Arthritis Ireland, received a questionnaire at the first and last week of the weekly ASMP. The questionnaire included the EC-17 and 10 other measures for arthritis. Deficits, mean change, and standard deviations were calculated at baseline and Week 6. The EC-17 scores were compared to the Arthritis Self-Efficacy (ASE) and Patient Activation Measure (PAM) scales. Results were presented at OMERACT 9. Results. There is some overlap between the EC-17 and the ASE and PAM; however, most items of greatest deficit in the EC-17 are not covered by those scales. In 327 participants representing both intervention waves (2006 and 2007), the EC-17 was more efficient than the ASE but less efficient than the PAM for detecting improvements after the ASMP, and was moderately correlated with the PAM. Conclusion. The EC-17 appears to measure different skills and attributes than the ASE and PAM. Discussions with participants at OMERACT 9 agreed that it is worthwhile to measure the skills and attributes of an effective consumer, and supported the development of an intervention (such as proposed online decision aids) that would include education in the categories in the EC-17.
Resumo:
Physical inactivity is a serious concern both nationally and internationally. Despite the numerous benefits of performing regular physical activity, many individuals lead sedentary lifestyles. Of concern, though, is research showing that some population sub-groups are less likely to be active, such as parents of young children. Although there is a vast amount of research dedicated to understanding people.s physical activity-related behaviours, there is a paucity of research examining those factors that influence parental physical activity. More importantly, research applying theoretical models to understand physical activity decision-making among this at-risk population is limited. Given the current obesity epidemic, the decline in physical activity with parenthood, and the many social and health benefits associated with regular physical activity, it is important that adults with young children are sufficiently active. In light of the dearth of research examining parental physical activity and the scant research applying a theory-based approach to gain this understanding, the overarching aim of the current program of research was to adopt a mixed methods approach as well as use sound theoretical frameworks to understand the regular physical activity behaviour of mothers and fathers with young children. This program of research comprised of three distinct stages: a qualitative stage exploring individual, social, and psychological factors that influence parental regular physical activity (Stage 1); a quantitative stage identifying the important predictors of parental regular physical activity intentions and behaviour using sound theoretical frameworks and testing a single-item measure for assessing parental physical activity behaviour (Stage 2); and a qualitative stage exploring strategies for an intervention program aimed at increasing parental regular physical activity (Stage 3). As a thesis by publication, eight papers report the findings of this program of research; these papers are presented according to the distinct stages of investigation that guided this program of research. Stage One of the research program comprised a qualitative investigation using a focus group/interview methodology with parents of children younger than 5 years of age (N = 40; n = 21 mothers, n = 19 fathers) (Papers 1, 2, and 3). Drawing broadly on a social constructionist approach (Paper 1), thematic analytic methods revealed parents. understandings of physical activity (e.g., requires effort), patterns of physical activity-related behaviours (e.g., grab it when you can, declining physical activity habits), and how constructions of social role expectations might influence parents. physical activity decision making (e.g., creating an active family culture, guilt and selfishness). Drawing on the belief-based framework of the TPB (Paper 2), thematic content analytic methods revealed parents. commonly held beliefs about the advantages (e.g., improves parenting practices), disadvantages (e.g., interferes with commitments), barriers (e.g., time), and facilitators (e.g., social support) to performing regular physical activity. Parents. normative beliefs about social approval from important others or groups (e.g., spouse/partner) were also identified. Guided by theories of social support, Paper Three identified parents. perceptions about the specific social support dimensions that influence their physical activity decision making. Thematic content analysis identified instrumental (e.g., providing childcare, taking over chores), emotional (e.g., encouragement, companionship), and informational support (e.g., ideas and advice) as being important to the decision-making of parents in relation to their regular physical activity behaviour. The results revealed also that having support for being active is not straightforward (e.g., guilt-related issues inhibited the facilitative nature of social support for physical activity). Stage Two of the research program comprised a quantitative examination of parents. physical activity intentions and behaviour (Papers 4, 5, 6, and 7). Parents completed an extended TPB questionnaire at Time 1 (N = 580; n = 288 mothers, n = 292 fathers) and self-reported their physical activity at Time 2, 1 week later (N = 458; n = 252 mothers, n = 206 fathers). Paper Four revealed key behavioural (e.g., improving parenting practices), normative (e.g., people I exercise with), and control (e.g., lack of time) beliefs as significant independent predictors of parental physical activity. A test of the TPB augmented to include the constructs of self-determined motivation and planning was assessed in Paper Five. The findings revealed that the effect of self-determined motivation on intention was fully mediated by the TPB variables and the impact of intention on behaviour was partially mediated by the planning variables. Slight differences in the model.s motivational sequence between the sexes were also noted. Paper Six investigated, within a TPB framework, a range of social influences on parents. intentions to be active. For both sexes, attitude, perceived behavioural control, group norms, friend general support, and an active parent identity predicted intentions, with subjective norms and family support further predicting mothers. intentions and descriptive norms further predicting fathers. intentions. Finally, the measurement of parental physical activity was investigated in Paper Seven of Stage Two. The results showed that parents are at risk of low levels of physical activity, with the findings also revealing validation support for a brief single-item physical activity measure. Stage Three of the research program comprised a qualitative examination of parents. (N = 12; n = 6 mothers, n = 6 fathers) ideas for strategies that may be useful for developing and delivering an intervention program aimed at increasing parental physical activity (Paper 8). Parents revealed a range of strategies for what to include in a physical activity intervention designed for parents of young children. For example, parents identified persuasion and information type messages, problem-solving strategies that engage parents in generating a priority list of their lifestyle commitments, and behavioural modification techniques such as goal setting and incentives. Social intervention strategies (e.g., social comparison, counselling) and environmental approaches (e.g., community-based integrative parent/child programs) were also identified as was a skill-based strategy in helping parents generate a flexible life/family plan. Additionally, a range of strategies for how to best deliver a parental physical activity intervention was discussed. Taken as a whole, Paper Eight found that adopting a multifaceted approach in both the design and implementation of a resultant physical activity intervention may be useful in helping to increase parental physical activity. Overall, this program of research found support for parents as a unique group who hold both similar and distinctive perceptions about regular physical activity to the general adult population. Thus, these findings highlight the importance of targeting intervention strategies for parents of young children. Additionally, the findings suggest that it might also be useful to tailor some messages specifically to each sex. Effective promotion of physical activity in parents of young children is essential given the low rate of activity in this population. Results from this program of research highlight parents as an at-risk group for inactivity and provide an important first step in identifying the factors that influence both mothers. and fathers. physical activity decision making. These findings, in turn, provide a foundation on which to build effective intervention programs aimed at increasing parents. regular physical activity which is essential for ensuring the health and well-being of parents with young children.
Resumo:
Demands for delivering high instantaneous power in a compressed form (pulse shape) have widely increased during recent decades. The flexible shapes with variable pulse specifications offered by pulsed power have made it a practical and effective supply method for an extensive range of applications. In particular, the release of basic subatomic particles (i.e. electron, proton and neutron) in an atom (ionization process) and the synthesizing of molecules to form ions or other molecules are among those reactions that necessitate large amount of instantaneous power. In addition to the decomposition process, there have recently been requests for pulsed power in other areas such as in the combination of molecules (i.e. fusion, material joining), gessoes radiations (i.e. electron beams, laser, and radar), explosions (i.e. concrete recycling), wastewater, exhausted gas, and material surface treatments. These pulses are widely employed in the silent discharge process in all types of materials (including gas, fluid and solid); in some cases, to form the plasma and consequently accelerate the associated process. Due to this fast growing demand for pulsed power in industrial and environmental applications, the exigency of having more efficient and flexible pulse modulators is now receiving greater consideration. Sensitive applications, such as plasma fusion and laser guns also require more precisely produced repetitive pulses with a higher quality. Many research studies are being conducted in different areas that need a flexible pulse modulator to vary pulse features to investigate the influence of these variations on the application. In addition, there is the need to prevent the waste of a considerable amount of energy caused by the arc phenomena that frequently occur after the plasma process. The control over power flow during the supply process is a critical skill that enables the pulse supply to halt the supply process at any stage. Different pulse modulators which utilise different accumulation techniques including Marx Generators (MG), Magnetic Pulse Compressors (MPC), Pulse Forming Networks (PFN) and Multistage Blumlein Lines (MBL) are currently employed to supply a wide range of applications. Gas/Magnetic switching technologies (such as spark gap and hydrogen thyratron) have conventionally been used as switching devices in pulse modulator structures because of their high voltage ratings and considerably low rising times. However, they also suffer from serious drawbacks such as, their low efficiency, reliability and repetition rate, and also their short life span. Being bulky, heavy and expensive are the other disadvantages associated with these devices. Recently developed solid-state switching technology is an appropriate substitution for these switching devices due to the benefits they bring to the pulse supplies. Besides being compact, efficient, reasonable and reliable, and having a long life span, their high frequency switching skill allows repetitive operation of pulsed power supply. The main concerns in using solid-state transistors are the voltage rating and the rising time of available switches that, in some cases, cannot satisfy the application’s requirements. However, there are several power electronics configurations and techniques that make solid-state utilisation feasible for high voltage pulse generation. Therefore, the design and development of novel methods and topologies with higher efficiency and flexibility for pulsed power generators have been considered as the main scope of this research work. This aim is pursued through several innovative proposals that can be classified under the following two principal objectives. • To innovate and develop novel solid-state based topologies for pulsed power generation • To improve available technologies that have the potential to accommodate solid-state technology by revising, reconfiguring and adjusting their structure and control algorithms. The quest to distinguish novel topologies for a proper pulsed power production was begun with a deep and through review of conventional pulse generators and useful power electronics topologies. As a result of this study, it appears that efficiency and flexibility are the most significant demands of plasma applications that have not been met by state-of-the-art methods. Many solid-state based configurations were considered and simulated in order to evaluate their potential to be utilised in the pulsed power area. Parts of this literature review are documented in Chapter 1 of this thesis. Current source topologies demonstrate valuable advantages in supplying the loads with capacitive characteristics such as plasma applications. To investigate the influence of switching transients associated with solid-state devices on rise time of pulses, simulation based studies have been undertaken. A variable current source is considered to pump different current levels to a capacitive load, and it was evident that dissimilar dv/dts are produced at the output. Thereby, transient effects on pulse rising time are denied regarding the evidence acquired from this examination. A detailed report of this study is given in Chapter 6 of this thesis. This study inspired the design of a solid-state based topology that take advantage of both current and voltage sources. A series of switch-resistor-capacitor units at the output splits the produced voltage to lower levels, so it can be shared by the switches. A smart but complicated switching strategy is also designed to discharge the residual energy after each supply cycle. To prevent reverse power flow and to reduce the complexity of the control algorithm in this system, the resistors in common paths of units are substituted with diode rectifiers (switch-diode-capacitor). This modification not only gives the feasibility of stopping the load supply process to the supplier at any stage (and consequently saving energy), but also enables the converter to operate in a two-stroke mode with asymmetrical capacitors. The components’ determination and exchanging energy calculations are accomplished with respect to application specifications and demands. Both topologies were simply modelled and simulation studies have been carried out with the simplified models. Experimental assessments were also executed on implemented hardware and the approaches verified the initial analysis. Reports on details of both converters are thoroughly discussed in Chapters 2 and 3 of the thesis. Conventional MGs have been recently modified to use solid-state transistors (i.e. Insulated gate bipolar transistors) instead of magnetic/gas switching devices. Resistive insulators previously used in their structures are substituted by diode rectifiers to adjust MGs for a proper voltage sharing. However, despite utilizing solid-state technology in MGs configurations, further design and control amendments can still be made to achieve an improved performance with fewer components. Considering a number of charging techniques, resonant phenomenon is adopted in a proposal to charge the capacitors. In addition to charging the capacitors at twice the input voltage, triggering switches at the moment at which the conducted current through switches is zero significantly reduces the switching losses. Another configuration is also introduced in this research for Marx topology based on commutation circuits that use a current source to charge the capacitors. According to this design, diode-capacitor units, each including two Marx stages, are connected in cascade through solid-state devices and aggregate the voltages across the capacitors to produce a high voltage pulse. The polarity of voltage across one capacitor in each unit is reversed in an intermediate mode by connecting the commutation circuit to the capacitor. The insulation of input side from load side is provided in this topology by disconnecting the load from the current source during the supply process. Furthermore, the number of required fast switching devices in both designs is reduced to half of the number used in a conventional MG; they are replaced with slower switches (such as Thyristors) that need simpler driving modules. In addition, the contributing switches in discharging paths are decreased to half; this decrease leads to a reduction in conduction losses. Associated models are simulated, and hardware tests are performed to verify the validity of proposed topologies. Chapters 4, 5 and 7 of the thesis present all relevant analysis and approaches according to these topologies.
Resumo:
Complex networks have been studied extensively due to their relevance to many real-world systems such as the world-wide web, the internet, biological and social systems. During the past two decades, studies of such networks in different fields have produced many significant results concerning their structures, topological properties, and dynamics. Three well-known properties of complex networks are scale-free degree distribution, small-world effect and self-similarity. The search for additional meaningful properties and the relationships among these properties is an active area of current research. This thesis investigates a newer aspect of complex networks, namely their multifractality, which is an extension of the concept of selfsimilarity. The first part of the thesis aims to confirm that the study of properties of complex networks can be expanded to a wider field including more complex weighted networks. Those real networks that have been shown to possess the self-similarity property in the existing literature are all unweighted networks. We use the proteinprotein interaction (PPI) networks as a key example to show that their weighted networks inherit the self-similarity from the original unweighted networks. Firstly, we confirm that the random sequential box-covering algorithm is an effective tool to compute the fractal dimension of complex networks. This is demonstrated on the Homo sapiens and E. coli PPI networks as well as their skeletons. Our results verify that the fractal dimension of the skeleton is smaller than that of the original network due to the shortest distance between nodes is larger in the skeleton, hence for a fixed box-size more boxes will be needed to cover the skeleton. Then we adopt the iterative scoring method to generate weighted PPI networks of five species, namely Homo sapiens, E. coli, yeast, C. elegans and Arabidopsis Thaliana. By using the random sequential box-covering algorithm, we calculate the fractal dimensions for both the original unweighted PPI networks and the generated weighted networks. The results show that self-similarity is still present in generated weighted PPI networks. This implication will be useful for our treatment of the networks in the third part of the thesis. The second part of the thesis aims to explore the multifractal behavior of different complex networks. Fractals such as the Cantor set, the Koch curve and the Sierspinski gasket are homogeneous since these fractals consist of a geometrical figure which repeats on an ever-reduced scale. Fractal analysis is a useful method for their study. However, real-world fractals are not homogeneous; there is rarely an identical motif repeated on all scales. Their singularity may vary on different subsets; implying that these objects are multifractal. Multifractal analysis is a useful way to systematically characterize the spatial heterogeneity of both theoretical and experimental fractal patterns. However, the tools for multifractal analysis of objects in Euclidean space are not suitable for complex networks. In this thesis, we propose a new box covering algorithm for multifractal analysis of complex networks. This algorithm is demonstrated in the computation of the generalized fractal dimensions of some theoretical networks, namely scale-free networks, small-world networks, random networks, and a kind of real networks, namely PPI networks of different species. Our main finding is the existence of multifractality in scale-free networks and PPI networks, while the multifractal behaviour is not confirmed for small-world networks and random networks. As another application, we generate gene interactions networks for patients and healthy people using the correlation coefficients between microarrays of different genes. Our results confirm the existence of multifractality in gene interactions networks. This multifractal analysis then provides a potentially useful tool for gene clustering and identification. The third part of the thesis aims to investigate the topological properties of networks constructed from time series. Characterizing complicated dynamics from time series is a fundamental problem of continuing interest in a wide variety of fields. Recent works indicate that complex network theory can be a powerful tool to analyse time series. Many existing methods for transforming time series into complex networks share a common feature: they define the connectivity of a complex network by the mutual proximity of different parts (e.g., individual states, state vectors, or cycles) of a single trajectory. In this thesis, we propose a new method to construct networks of time series: we define nodes by vectors of a certain length in the time series, and weight of edges between any two nodes by the Euclidean distance between the corresponding two vectors. We apply this method to build networks for fractional Brownian motions, whose long-range dependence is characterised by their Hurst exponent. We verify the validity of this method by showing that time series with stronger correlation, hence larger Hurst exponent, tend to have smaller fractal dimension, hence smoother sample paths. We then construct networks via the technique of horizontal visibility graph (HVG), which has been widely used recently. We confirm a known linear relationship between the Hurst exponent of fractional Brownian motion and the fractal dimension of the corresponding HVG network. In the first application, we apply our newly developed box-covering algorithm to calculate the generalized fractal dimensions of the HVG networks of fractional Brownian motions as well as those for binomial cascades and five bacterial genomes. The results confirm the monoscaling of fractional Brownian motion and the multifractality of the rest. As an additional application, we discuss the resilience of networks constructed from time series via two different approaches: visibility graph and horizontal visibility graph. Our finding is that the degree distribution of VG networks of fractional Brownian motions is scale-free (i.e., having a power law) meaning that one needs to destroy a large percentage of nodes before the network collapses into isolated parts; while for HVG networks of fractional Brownian motions, the degree distribution has exponential tails, implying that HVG networks would not survive the same kind of attack.
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Is it possible to control identities using performance management systems (PMSs)? This paper explores the theoretical fusion of management accounting and identity studies, providing a synthesised view of control, PMSs and identification processes. It argues that the effective use of PMSs generates a range of obtrusive mechanistic and unobtrusive organic controls that mediate identification processes to achieve a high level of identity congruency between individuals and collectives—groups and organisations. This paper contends that mechanistic control of PMSs provides sensebreaking effects and also creates structural conditions for sensegiving in top-down identification processes. These processes encourage individuals to continue the bottom-up processes of sensemaking, enacting identity and constructing identity narratives. Over time, PMS activities and conversations periodically mediate several episode(s) of identification to connect past, current and future identities. To explore this relationship, the dual locus of control—collectives and individuals—is emphasised to explicate their interplay. This multidisciplinary approach contributes to explaining the multidirectional effects of PMSs in obtrusive as well as unobtrusive ways, in order to control the nature of collectives and individuals in organisations.
Resumo:
Is it possible to control identities using performance management systems (PMSs)? This paper explores the theoretical fusion of management accounting and identity studies, providing a synthesised view of control, PMSs and identification processes. It argues that the effective use of PMSs generates a range of obtrusive mechanistic and unobtrusive organic controls that mediate identification processes to achieve a high level of identity congruency between individuals and collectives—groups and organisations. This paper contends that mechanistic control of PMSs provides sensebreaking effects and also creates structural conditions for sensegiving in top-down identification processes. These processes encourage individuals to continue the bottom-up processes of sensemaking, enacting identity and constructing identity narratives. Over time, PMS activities and conversations periodically mediate several episode(s) of identification to connect past, current and future identities. To explore this relationship, the dual locus of control—collectives and individuals—is emphasised to explicate their interplay. This multidisciplinary approach contributes to explaining the multidirectional effects of PMSs in obtrusive as well as unobtrusive ways, in order to control the nature of collectives and individuals in organisations.
Using cost-effective multimedia to create engaging learning experiences in law and other disciplines
Resumo:
This is the final report of an Australian Learning and Teaching Council Teaching Fellowship which addressed the needs of two separate groups of learners: (1) final year law students studying ethics and (2) law academics and other interested educators in higher education wishing to use information and communication technologies (ICT) to create engaging learning environments for their students but lacking the capacity to do so. The Fellowship resulted in final year law students being infused with an improved appreciation of ethical practice than they receive from traditional lecture/tutorial means by the development of an integrated program of blended learning including an online program entitled "Entry into Valhalla". This "ethics capstone‟ utilises multimedia produced using cost effective resources (including the "Second Life" virtual environment) to create engaging, contextualised learning experiences. The Fellowship also constructed the knowledge of producing cost-effective multimedia projects in other law academics and other educators in higher education by staff development activities comprising workshops, conference presentations and an interactive website using the "Entry into Valhalla" program as a case study exemplar.
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Audience Response Systems (ARS) have been successfully used by academics to facilitate student learning and engagement, particularly in large lecture settings. However, in large core subjects a key challenge is not only to engage students, but also to engage large and diverse teaching teams in order to ensure a consistent approach to grading assessments. This paper provides an insight into the ways in which ARS can be used to encourage participation by tutors in marking and moderation meetings. It concludes that ARS can improve the consistency of grading and the quality of feedback provided to students.
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Research Interests: Are parents complying with the legislation? Is this the same for urban, regional and rural parents? Indigenous parents? What difficulties do parents experience in complying? Do parents understand why the legislation was put in place? Have there been negative consequences for other organisations or sectors of the community?
Resumo:
Part-time employment presents a conundrum in that it facilitates work-life priorities, while also, compared to equivalent full-time roles, attracting penalties such as diminished career prospects and lower commensurate remuneration. Recently, some promising theoretical developments in the job/work design literature suggest that consideration of work design may redress some of the penalties associated with part-time work. Adopting the framework of the Elaborated Model of Work Design by Parker and colleagues (2001), we examined this possibility through interviews with part-time professional service employees and their supervisors. The findings revealed that in organizations characterised by cultural norms of extended working hours and a singular-focused commitment to work, part-time roles were often inadequately re-designed when adapted from full-time arrangements. The findings also demonstrated that certain work design characteristics (e.g. predictability of work-flow, interdependencies with co-workers) render some roles more suitable for part-time arrangements than others. The research provides insights into gaps between policy objectives and outcomes associated with part-time work, challenges assumptions about the limitations of part-time roles, and suggests re-design strategies for more effective part-time arrangements.
Resumo:
Vitamin A deficiency (VAD) is a serious problem in developing countries, affecting approximately 127 million children of preschool age and 7.2 million pregnant women each year. However, this deficiency is readily treated and prevented through adequate nutrition. This can potentially be achieved through genetically engineered biofortification of staple food crops to enhance provitamin A (pVA) carotenoid content. Bananas are the fourth most important food crop with an annual production of 100 million tonnes and are widely consumed in areas affected by VAD. However, the fruit pVA content of most widely consumed banana cultivars is low (~ 0.2 to 0.5 ìg/g dry weight). This includes cultivars such as the East African highland banana (EAHB), the staple crop in countries such as Uganda, where annual banana consumption is approximately 250 kg per person. This fact, in addition to the agronomic properties of staple banana cultivars such as vegetative reproduction and continuous cropping, make bananas an ideal target for pVA enhancement through genetic engineering. Interestingly, there are banana varieties known with high fruit pVA content (up to 27.8 ìg/g dry weight), although they are not widely consumed due to factors such as cultural preference and availability. The genes involved in carotenoid accumulation during banana fruit ripening have not been well studied and an understanding of the molecular basis for the differential capacity of bananas to accumulate carotenoids may impact on the effective production of genetically engineered high pVA bananas. The production of phytoene by the enzyme phytoene synthase (PSY) has been shown to be an important rate limiting determinant of pVA accumulation in crop systems such as maize and rice. Manipulation of this gene in rice has been used successfully to produce Golden Rice, which exhibits higher seed endosperm pVA levels than wild type plants. Therefore, it was hypothesised that differences between high and low pVA accumulating bananas could be due either to differences in PSY enzyme activity or factors regulating the expression of the psy gene. Therefore, the aim of this thesis was to investigate the role of PSY in accumulation of pVA in banana fruit of representative high (Asupina) and low (Cavendish) pVA banana cultivars by comparing the nucleic acid and encoded amino acid sequences of the banana psy genes, in vivo enzyme activity of PSY in rice callus and expression of PSY through analysis of promoter activity and mRNA levels. Initially, partial sequences of the psy coding region from five banana cultivars were obtained using reverse transcriptase (RT)-PCR with degenerate primers designed to conserved amino acids in the coding region of available psy sequences from other plants. Based on phylogenetic analysis and comparison to maize psy sequences, it was found that in banana, psy occurs as a gene family of at least three members (psy1, psy2a and psy2b). Subsequent analysis of the complete coding regions of these genes from Asupina and Cavendish suggested that they were all capable of producing functional proteins due to high conservation in the catalytic domain. However, inability to obtain the complete mRNA sequences of Cavendish psy2a, and isolation of two non-functional Cavendish psy2a coding region variants, suggested that psy2a expression may be impaired in Cavendish. Sequence analysis indicated that these Cavendish psy2a coding region variants may have resulted from alternate splicing. Evidence of alternate splicing was also observed in one Asupina psy1 coding region variant, which was predicted to produce a functional PSY1 isoform. The complete mRNA sequence of the psy2b coding regions could not be isolated from either cultivar. Interestingly, psy1 was cloned predominantly from leaf while psy2 was obtained preferentially from fruit, suggesting some level of tissue-specific expression. The Asupina and Cavendish psy1 and psy2a coding regions were subsequently expressed in rice callus and the activity of the enzymes compared in vivo through visual observation and quantitative measurement of carotenoid accumulation. The maize B73 psy1 coding region was included as a positive control. After several weeks on selection, regenerating calli showed a range of colours from white to dark orange representing various levels of carotenoid accumulation. These results confirmed that the banana psy coding regions were all capable of producing functional enzymes. No statistically significant differences in levels of activity were observed between banana PSYs, suggesting that differences in PSY activity were not responsible for differences in the fruit pVA content of Asupina and Cavendish. The psy1 and psy2a promoter sequences were isolated from Asupina and Cavendish gDNA using a PCR-based genome walking strategy. Interestingly, three Cavendish psy2a promoter clones of different sizes, representing possible allelic variants, were identified while only single promoter sequences were obtained for the other Asupina and Cavendish psy genes. Bioinformatic analysis of these sequences identified motifs that were previously characterised in the Arabidopsis psy promoter. Notably, an ATCTA motif associated with basal expression in Arabidopsis was identified in all promoters with the exception of two of the Cavendish psy2a promoter clones (Cpsy2apr2 and Cpsy2apr3). G1 and G2 motifs, linked to light-regulated responses in Arabidopsis, appeared to be differentially distributed between psy1 and psy2a promoters. In the untranscribed regulatory regions, the G1 motifs were found only in psy1 promoters, while the G2 motifs were found only in psy2a. Interestingly, both ATCTA and G2 motifs were identified in the 5’ UTRs of Asupina and Cavendish psy1. Consistent with other monocot promoters, introns were present in the Asupina and Cavendish psy1 5’ UTRs, while none were observed in the psy2a 5’ UTRs. Promoters were cloned into expression constructs, driving the â-glucuronidase (GUS) reporter gene. Transient expression of the Asupina and Cavendish psy1 and psy2a promoters in both Cavendish embryogenic cells and Cavendish fruit demonstrated that all promoters were active, except Cpsy2apr2 and Cpsy2apr3. The functional Cavendish psy2a promoter (Cpsy2apr1) appeared to have activity similar to the Asupina psy2a promoter. The activities of the Asupina and Cavendish psy1 promoters were similar to each other, and comparable to those of the functional psy2a promoters. Semi-quantitative PCR analysis of Asupina and Cavendish psy1 and psy2a transcripts showed that psy2a levels were high in green fruit and decreased during ripening, reinforcing the hypothesis that fruit pVA levels were largely dependent on levels of psy2a expression. Additionally, semi-quantitative PCR using intron-spanning primers indicated that high levels of unprocessed psy2a and psy2b mRNA were present in the ripe fruit of Cavendish but not in Asupina. This raised the possibility that differences in intron processing may influence pVA accumulation in Asupina and Cavendish. In this study the role of PSY in banana pVA accumulation was analysed at a number of different levels. Both mRNA accumulation and promoter activity of psy genes studied were very similar between Asupina and Cavendish. However, in several experiments there was evidence of cryptic or alternate splicing that differed in Cavendish compared to Asupina, although these differences were not conclusively linked to the differences in fruit pVA accumulation between Asupina and Cavendish. Therefore, other carotenoid biosynthetic genes or regulatory mechanisms may be involved in determining pVA levels in these cultivars. This study has contributed to an increased understanding of the role of PSY in the production of pVA carotenoids in banana fruit, corroborating the importance of this enzyme in regulating carotenoid production. Ultimately, this work may serve to inform future research into pVA accumulation in important crop varieties such as the EAHB and the discovery of avenues to improve such crops through genetic modification.