The manipulation of apoptosis-related genes to generate resistance to Fusarium wilt and water stress in banana

Bananas are susceptible to a diverse range of biotic and abiotic stresses, many of which cause serious production constraints worldwide. One of the most destructive banana diseases is Fusarium wilt caused by the soil-borne fungus, Fusarium oxysporum f. sp. cubense (Foc). No effective control strategy currently exists for this disease which threatens global banana production. Although disease resistance exists in some wild bananas, attempts to introduce resistance into commercially acceptable bananas by conventional breeding have been hampered by low fertility, long generation times and association of poor agronomical traits with resistance genes. With the advent of reliable banana transformation protocols, molecular breeding is now regarded as a viable alternative strategy to generate disease-resistant banana plants. Recently, a novel strategy involving the expression of anti-apoptosis genes in plants was shown to result in resistance against several necrotrophic fungi. Further, the transgenic plants showed increased resistance to a range of abiotic stresses. In this thesis, the use of anti-apoptosis genes to generate transgenic banana plants with resistance to Fusarium wilt was investigated. Since water stress is an important abiotic constraint to banana production, the resistance of the transgenic plants to water stress was also examined. Embryogenic cell suspensions (ECS) of two commercially important banana cultivars, Grand Naine (GN) and Lady Finger (LF), were transformed using Agrobacterium with the anti-apoptosis genes, Bcl-xL, Bcl-xL G138A, Ced-9 and Bcl- 2 3’ UTR. An interesting, and potentially important, outcome was that the use of anti-apoptosis genes resulted in up to a 50-fold increase in Agrobacterium-mediated transformation efficiency of both LF and GN cells over vector controls. Regenerated plants were subjected to a complete molecular characterisation in order to detect the presence of the transgene (PCR), transcript (RT-PCR) and gene product (Western blot) and to determine the gene copy number (Southern blot). A total of 36 independently-transformed GN lines (8 x Bcl-xL, 5 x Bcl-xL G138A, 15 x Ced-9 and 8 x Bcl-2 3’ UTR) and 41 independently-transformed LF lines (8 x Bcl-xL, 7 x BclxL G138A, 13 x Ced-9 and 13 x Bcl-2 3’ UTR) were identified. The 41 transgenic LF lines were multiplied and clones from each line were acclimatised and grown under glasshouse conditions for 8 weeks to allow monitoring for phenotypic abnormalities. Plants derived from 3 x Bcl-xL, 2 x Ced-9 and 5 x Bcl-2 3’ UTR lines displayed a variety of aberrant phenotypes. However, all but one of these abnormalities were off-types commonly observed in tissue-cultured, non-transgenic banana plants and were therefore unlikely to be transgene-related. Prior to determining the resistance of the transgenic plants to Foc race 1, the apoptotic effects of the fungus on both wild-type and Bcl-2 3’ UTR-transgenic LF banana cells were investigated using rapid in vitro root assays. The results from these assays showed that apoptotic-like cell death was elicited in wild-type banana root cells as early as 6 hours post-exposure to fungal spores. In contrast, these effects were attenuated in the root cells of Bcl-2 3’ UTR-transgenic lines that were exposed to fungal spores. Thirty eight of the 41 transgenic LF lines were subsequently assessed for resistance to Foc race 1 in small-plant glasshouse bioassays. To overcome inconsistencies in rating the internal (vascular discolouration) disease symptoms, a MatLab-based computer program was developed to accurately and reliably assess the level of vascular discolouration in banana corms. Of the transgenic LF banana lines challenged with Foc race 1, 2 x Bcl-xL, 3 x Ced-9, 2 x Bcl-2 3’ UTR and 1 x Bcl-xL G138A-transgenic line were found to show significantly less external and internal symptoms than wild-type LF banana plants used as susceptible controls at 12 weeks post-inoculation. Of these lines, Bcl-2 3’ UTR-transgenic line #6 appeared most resistant, displaying very mild symptoms similar to the wild-type Cavendish banana plants that were included as resistant controls. This line remained resistant for up to 23 weeks post-inoculation. Since anti-apoptosis genes have been shown to confer resistance to various abiotic stresses in other crops, the ability of these genes to confer resistance against water stress in banana was also investigated. Clonal plants derived from each of the 38 transgenic LF banana plants were subjected to water stress for a total of 32 days. Several different lines of transgenic plants transformed with either Bcl-xL, Bcl-xL G138A, Ced-9 or Bcl-2 3’ UTR showed a delay in visual water stress symptoms compared with the wild-type control plants. These plants all began producing new growth from the pseudostem following daily rewatering for one month. In an attempt to determine whether the protective effect of anti-apoptosis genes in transgenic banana plants was linked with reactive oxygen species (ROS)-associated programmed cell death (PCD), the effect of the chloroplast-targeting, ROS-inducing herbicide, Paraquat, on wild-type and transgenic LF was investigated. When leaf discs from wild-type LF banana plants were exposed to 10 ìM Paraquat, complete decolourisation occurred after 48 hours which was confirmed to be associated with cell death and ROS production by trypan blue and 3,3-diaminobenzidine (DAB) staining, respectively. When leaf discs from the transgenic lines were exposed to Paraquat, those derived from some lines showed a delay in decolourisation, suggesting only a weak protective effect from the transgenes. Finally, the protective effect of anti-apoptosis genes against juglone, a ROS-inducing phytotoxin produced by the causal agent of black Sigatoka, Mycosphaerella fijiensis, was investigated. When leaf discs from wild-type LF banana plants were exposed to 25 ppm juglone, complete decolourisation occurred after 48 hours which was again confirmed to be associated with cell death and ROS production by trypan blue and DAB staining, respectively. Further, TdT-mediated dUTP nick-end labelling (TUNEL) assays on these discs suggested that the cell death was apoptotic. When leaf discs from the transgenic lines were exposed to juglone, discs from some lines showed a clear delay in decolourisation, suggesting a protective effect. Whether these plants are resistant to black Sigatoka is unknown and will require future glasshouse and field trials. The work presented in this thesis provides the first report of the use of anti-apoptosis genes as a strategy to confer resistance to Fusarium wilt and water stress in a nongraminaceous monocot, banana. Such a strategy may be exploited to generate resistance to necrotrophic pathogens and abiotic stresses in other economically important crop plants.

Early objective detection of lymphoedema

The aetiology of secondary lymphoedema seems to be multifactorial, with acquired abnormalities as well as pre-existing conditions being contributory factors. Many characteristics bear inconsistent relationships to lymphoedema risk, and the few that are consistently associated with an increased risk of developing the condition, do not alone distinguish the at-risk population. Further, our current prevention and management recommendations are not backed by strong evidence. Consequently, there remains much to be learned about who gets it, how can it be prevented and how can we best treat it. Nonetheless, it is clear that lymphoedema is associated with adverse side effects, which have a profound impact on daily life, and that preliminary evidence suggests that early detection may lead to more effective treatment and lack of treatment may lead to progression. These represent important reasons as to why lymphoedema deserves clinical attention. However, several pragmatic issues must be considered when discussing whether a routine objective measure of lymphoedema could be integrated among the standard clinical care of those undertaking treatment for cancers known to be associated with the development of lymphoedema.

Minimising speaker verification utterance length through confidence based early verification decisions

This paper presents a novel approach of estimating the confidence interval of speaker verification scores. This approach is utilised to minimise the utterance lengths required in order to produce a confident verification decision. The confidence estimation method is also extended to address both the problem of high correlation in consecutive frame scores, and robustness with very limited training samples. The proposed technique achieves a drastic reduction in the typical data requirements for producing confident decisions in an automatic speaker verification system. When evaluated on the NIST 2005 SRE, the early verification decision method demonstrates that an average of 5–10 seconds of speech is sufficient to produce verification rates approaching those achieved previously using an average in excess of 100 seconds of speech.

Is liraglutide or exenatide better in type 2 diabetes?

Background: Subjects with type 2 diabetes have high circulating levels of glucose. Glucagon-like peptide-1 (GLP-1) is an intestinal hormone that has a major role in glucose homeostasis. Exenatide and liraglutide are both agonists at the GLP-1 receptor, and are effective at reducing circulating glucose levels (measured as HbA1c levels), but they have not been compared. Objectives/methods: This evaluation is of a clinical trial comparing liraglutide once a day with exenatide twice a day in subjects with type 2 diabetes. Results: In the Liraglutide Effect and Action in Diabetes (LEAD)-6 trial, subcutaneous liraglutide 1.8 mg once a day was compared with exenatide 10 μg twice a day. The primary efficacy outcome was change in HbA1c levels, and this was significantly greater with liraglutide (1.12%) than with exenatide (0.79%). Liraglutide and exenatide had similar small abilities to reduce body weight, blood pressure and LDL-cholesterol. Conclusions: Liraglutide was more effective than exenatide for overall glycaemic control in subjects with type 2 diabetes. However, this is only true for the preparations and doses tested, that is liraglutide 1.8 mg once weekly and exenatide 10 μg b.i.d., and may not apply when the comparison is undertaken with the new longer-lasting preparation of exenatide once weekly.

Early outcomes of patella resurfacing in total knee arthroplasty

Background Patella resurfacing in total knee arthroplasty is a contentious issue. The literature suggests that resurfacing of the patella is based on surgeon preference, and little is known about the role and timing of resurfacing and how this affects outcomes. Methods We analyzed 134,799 total knee arthroplasties using data from the Australian Orthopaedic Association National Joint Replacement Registry. Hazards ratios (HRs) were used to compare rates of early revision between patella resurfacing at the primary procedure (the resurfacing group, R) and primary arthroplasty without resurfacing (no-resurfacing group, NR). We also analyzed the outcomes of NR that were revised for isolated patella addition. Results At 5 years, the R group showed a lower revision rate than the NR group: cumulative per cent revision (CPR) 3.1% and 4.0%, respectively (HR = 0.75, p < 0.001). Revisions for patellofemoral pain were more common in the NR group (17%) than in the R group (1%), and “patella only” revisions were more common in the NR group (29%) than in the R group (6%). Non-resurfaced knees revised for isolated patella addition had a higher revision rate than patella resurfacing at the primary procedure, with a 4-year CPR of 15% and 2.8%, respectively (HR = 4.1, p < 0.001). Interpretation Rates of early revision of primary total knees were higher when the patella was not resurfaced, and suggest that surgeons may be inclined to resurface later if there is patellofemoral pain. However, 15% of non-resurfaced knees revised for patella addition are re-revised by 4 years. Our results suggest an early beneficial outcome for patella resurfacing at primary arthroplasty based on revision rates up to 5 years.

Ecological perspectives and wellness

Since the industrial revolution, the development of a lifestyle lived predominantly indoors has resulted in less contact with nature. Research over the last twenty years has gradually been identifying the human health benefits attributed to re-connecting with the natural environment. The significance of feeling connected to natural environments, families and friends are described as a foundational requirement for human health and wellbeing (Maller et al., 2008). Also, the early findings of Schultz‟s (2002) work indicated that by feeling connected to the natural world a person is more likely to be committed to positively interact with and protect the natural world. Research on young people has indicated that young people are even more disconnected from the natural world. Leading some writers to call this disconnection a crisis termed “Nature Deficit Disorder.” Participants (n = 131) from 1st year university Physical Education and Human Movement Studies were asked to complete two questionnaires the Connectedness to Nature scale (CNS) (Mayer & Frantz, 2004) and the New Ecological Paradigm Scale (NEP) (Dunlap, Van Liere, Mertig, & Jones, 2000). The NEP and CNS are two scales most commonly used to explore beliefs and feelings of connectedness to the natural world (Schultz, 2002). The NEP was developed over thirty years ago by Dunlap and Van Liere (1978) and originally termed the New Environmental Paradigm. The NEP is now the foremost International tool for measuring beliefs about the natural world (Dunlap, 2008). The CNS measures an individual‟s trait levels of emotional connection to the natural world. It is a relatively new tool for understanding ecological behaviour based on ecopsychology theory and employed to predict behaviour (Mayer and Frantz, 2004). Both questionnaires are based on a 1-5 scale (Strongly disagree to Strongly agree). By combing both scales the researchers aim to develop a snap shot of beliefs and emotional feelings towards the natural world and therefore an idea of intended behaviour. The two questionnaires were combined as one online survey with additional material asking for demographics and self assessments of type of leader included before the surveys. An email inviting outdoor leaders to participate was sent out to networks and interest groups. A basic descriptive statistical analysis was used to interpret data.

Social inequalities and parent involvement in children’s education in the early years of school

Strengthening cooperation between schools and parents is critical to improving learning outcomes for children. The chapter focuses on parental engagement in their children’s education in the early years of school. It considers issues of social and cultural capital as important to whether, or not, parents are involved in their children’s schooling. Analyses of data from a national representative sample of children and their families who participate in Growing up in Australia: The Longitudinal Study of Australian Children are presented. Results indicated that higher family socio-economic position was associated with higher levels of parental involvement and higher expectations about children’s future level of education.

Resistance training for obese, type 2 diabetic adults: A review of the evidence

In both developed and developing countries, increased prevalence of obesity has been strongly associated with increased incidence of type 2 diabetes mellitus (T2DM) in the adult population. Previous research has emphasized the importance of physical activity in the prevention and management of obesity and T2DM, and generic exercise guidelines originally developed for the wider population have been adapted for these specific populations. However, the guidelines traditionally focus on aerobic training without due consideration to other exercise modalities. Recent reviews on resistance training in the T2DM population have not compared this modality with others including aerobic training, or considered the implications of resistance training for individuals suffering from both obesity and T2DM. In short, the optimal mix of exercise modalities in the prescription of exercise has not been identified for it benefits to the metabolic, body composition and muscular health markers common in obesity and T2DM. Similarly, the underlying physical, social and psychological barriers to adopting and maintaining exercise, with the potential to undermine the efficacy of exercise interventions, have not been addressed in earlier reviews. Because it is well established that aerobic exercise has profound effects on obesity and T2DM risk, the purpose of this review was to address the importance of resistance training to obese adults with T2DM.

Nature's experiment? Handedness and early childhood development

In recent years a large body of research has investigated the various factors affecting child development and the consequent impact of child development on future educational and labour market outcomes. In this article we contribute to this literature by investigating the effect of handedness on a child and given recent research demonstrating that child development strongly affects adult outcomes. Using a large nationally representative sample of young children we find that the probability of a child being left-handed is not significantly related to child health at birth, family composition, parental employment or household income. We also find robust evidence that left-handed (and mixed handed) children perform significantly worse in nearly all measures of development than right-handed children with the relative disadvantage being larger for boys than girls. Importantly these differentials cannot be explained by different socioeconomic characteristics of the household, parental attitudes or investments in learning resources.

The nature and extent of policing alcohol related crime and reducing violence in and around late night entertainment areas

The misuse of alcohol is well documented in Australia and has been associated with disorders and harms that often require police attention. The extent of alcohol-related incidents requiring police attention has been recorded as substantial in some Australian cities (Arro, Crook, & Fenton, 1992; Davey & French, 1995; Ireland & Thommeny, 1993). A significant proportion of harmful drinking occurs in and around licensed premises (Jochelson, 1997; Stockwell, Masters, Phillips, Daly, Gahegan, Midford, & Philp, 1998; Borges, Cherpitel, & Rosovsky, 1998) and most of these incidents are not reported to police (Bryant & Williams, 2000; Lister, Hobbs, Hall, & Winlow, 2000). Alcohol-related incidents have also been found to be concentrated in certain places at certain times (Jochelson, 1997) and therefore manipulating the context in which these incidents occur may provide a means to prevent and reduce the harm associated with alcohol misuse. One of the major objectives of the present program of research was to investigate the occurrence and resource impact of alcohol-related incidents on operational (general duties) policing across a large geographical area. A second objective of the thesis was to examine the characteristics and temporal/spatial dynamics of police attended alcohol incidents in the context of Place Based theories of crime. It was envisaged that this approach would reveal the patterns of the most prevalent offences and demonstrate the relevance of Place Based theories of crime to understanding these patterns. In addition, the role of alcohol, time and place were also explored in order to examine the association between non criminal traffic offences and other types of criminal offences. A final objective of the thesis was to examine the impact of a situational crime prevention strategy that had been initiated to reduce the violence and disorder associated with late-night liquor trading premises. The program of research in this doctorate thesis has been undertaken through the presentation of published papers. The research was conducted in three stages which produced six manuscripts, five of which were submitted to peer reviewed journals and one that was published in a peer reviewed conference proceedings. Stage One included two studies (Studies 1 & 2) both of which involved a cross sectional approach to examine the prevalence and characteristics of alcohol-related incidents requiring police attendance across three large geographical areas that included metropolitan cities, provincial regions and rural areas. Stage Two of the program of research also comprised two cross sectional quantitative studies (Studies 3 & 4) that investigated the temporal and spatial dynamics of the major offence categories attended by operational police in a specific Police District (Gold Coast). Stage Three of the program of research involved two studies (Studies 5 & 6) that assessed the effectiveness of a situational crime prevention strategy. The studies employed a pre-post design to assess the impact on crime, disorder and violence by preventing patrons from entering late-night liquor trading premises between 3 a.m. and 5 a.m. (lockout policy). Although Study Five was solely quantitative in nature, Study Six included both quantitative and qualitative aspects. The approach adopted in Study Six, therefore facilitated not only a quantative comparison of the impact of the lockout policy on different policing areas, but also enabled the processes related to the implementation of the lockout policy to be examined. The thesis reports a program of research involving a common data collection method which then involved a series of studies being conducted to explore different aspects of the data. The data was collected from three sources. Firstly a pilot phase was undertaken to provide participants with training. Secondly a main study period was undertaken immediately following the pilot phase. The first and second sources of data were collected between 29th March 2004 and 2nd May 2004. Thirdly, additional data was collected between the 1st April 2005 and 31st May 2005. Participants in the current program of research were first response operational police officers who completed a modified activity log over a 9 week period (4 week pilot phase & 5 week survey study phase), identifying the type, prevalence and characteristics of alcohol-related incidents that were attended. During the study period police officers attended 31,090 alcohol-related incidents. Studies One and Two revealed that a substantial proportion of current police work involves attendance at alcohol-related incidents (i.e., 25% largely involving young males aged between 17 and 24 years). The most common incidents police attended were vehicle and/or traffic matters, disturbances and offences against property. The major category of offences most likely to involve alcohol included vehicle/traffic matters, disturbances and offences against the person (e.g., common & serious assaults). These events were most likely to occur in the late evenings and early hours of the morning on the weekends, and importantly, usually took longer for police to complete than non alcohol-related incidents. The findings in Studies Three and Four suggest that serious traffic offences, disturbances and offences against the person share similar characteristics and occur in concentrated places at similar times. In addition, it was found that time, place and incident type all have an influence on whether an incident attended by a police officer is alcohol-related. Alcohol-related incidents are more likely to occur in particular locations in the late evenings and early mornings on the weekends. In particular, there was a strong association between the occurrence of alcohol-related disturbances and alcohol-related serious traffic offences in regards to place and time. In general, stealing and property offences were not alcohol-related and occurred in daylight hours during weekdays. The results of Studies Five and Six were mixed. A number of alcohol-related offences requiring police attention were significantly reduced for some policing areas and for some types of offences following the implementation of the lockout policy. However, in some locations the lockout policy appeared to have a negative or minimal impact. Interviews with licensees revealed that although all were initially opposed to the lockout policy as they believed it would have a negative impact on business, most perceived some benefits from its introduction. Some of the benefits included, improved patron safety and the development of better business strategies to increase patron numbers. In conclusion, the overall findings of the six studies highlight the pervasive nature of alcohol across a range of criminal incidents, demonstrating the tremendous impact alcohol-related incidents have on police. The findings also demonstrate the importance of time and place in predicting the occurrence of alcohol-related offences. Although this program of research did not set out to test Place Based theories of crime, these theories were used to inform the interpretation of findings. The findings in the current research program provide evidence for the relevance of Place Based theories of crime to understanding the factors contributing to violence and disorder, and designing relevant crime prevention strategies. For instance, the results in Studies Five and Six provide supportive evidence that this novel lockout initiative can be beneficial for public safety by reducing some types of offences in particular areas in and around late-night liquor trading premises. Finally, intelligent-led policing initiatives based on problem oriented policing, such as the lockout policy examined in this thesis, have potential as a major crime prevention technique to reduce specific types of alcohol-related offences.

Type Test Elimination using Typeflow Analysis

Programs written in languages of the Oberon family usually contain runtime tests on the dynamic type of variables. In some cases it may be desirable to reduce the number of such tests. Typeflow analysis is a static method of determining bounds on the types that objects may possess at runtime. We show that this analysis is able to reduce the number of tests in certain plausible circumstances. Furthermore, the same analysis is able to detect certain program errors at compile time, which would normally only be detected at program execution. This paper introduces the concepts of typeflow analysis and details its use in the reduction of runtime overhead in Oberon-2.

User-designer collaboration during the early stage of the design process

As user involvement becomes a necessary part of the product development process, various ways of accessing users' latent needs have been developed and studied. Reviews of literatures in user involvement and product development have revealed that accessing users' latent needs and transferring them into design process could be facilitated by effectively implementing user-designer collaboration during the early stage of the design process. In this paper, various types of user-designer collaboration were observed and then distinct characteristics of user-designer collaboration were classified into three categories. 1) Passive objectivity, 2) workplace democratisation, and 3) shared contexts were observed as strategies for better user-designer collaboration, which have been employed in the area of user-centred design, user participatory design and design for experiencing. Based on the literature review, this paper proposed a basic collaboration mechanism between the users and the designers during the early stage of the design process and then discussed how its mechanism will help to describe the interactions between the users and the designers during the user involvement sessions.

Kinematics of intracellular chlamydiae provide evidence for contact-dependent development

A crucial process of chlamydial development involves differentiation of the replicative reticulate body (RB) into the infectious elementary body (EB). We present experimental evidence to provide support for a contact-dependent hypothesis for explaining the trigger involved in differentiation. We recorded live-imaging of Chlamydia trachomatis-infected McCoy cells at key times during development and tracked the temporospatial trajectories of individual chlamydial particles. We found that movement of the particles is related to development. Early to mid-developmental stages involved slight wobbling of RBs. The average speed of particles increased sharply at 24 h postinfection (after the estimated onset of RB to EB differentiation). We also investigated a penicillin-supplemented culture containing EBs, RBs, and aberrantly enlarged, stressed chlamydiae. Near-immobile enlarged particles are consistent with their continued tethering to the chlamydial inclusion membrane (CIM). We found a significantly negative, nonlinear association between speed and size/type of particles, providing further support for the hypothesis that particles become untethered near the onset of RB to EB differentiation. This study establishes the relationship between the motion properties of the chlamydiae and developmental stages, whereby wobbling RBs gradually lose contact with the CIM, and RB detachment from the CIM is coincidental with the onset of late differentiation.

Detecting and characterising malicious executable payloads

Buffer overflow vulnerabilities continue to prevail and the sophistication of attacks targeting these vulnerabilities is continuously increasing. As a successful attack of this type has the potential to completely compromise the integrity of the targeted host, early detection is vital. This thesis examines generic approaches for detecting executable payload attacks, without prior knowledge of the implementation of the attack, in such a way that new and previously unseen attacks are detectable. Executable payloads are analysed in detail for attacks targeting the Linux and Windows operating systems executing on an Intel IA-32 architecture. The execution flow of attack payloads are analysed and a generic model of execution is examined. A novel classification scheme for executable attack payloads is presented which allows for characterisation of executable payloads and facilitates vulnerability and threat assessments, and intrusion detection capability assessments for intrusion detection systems. An intrusion detection capability assessment may be utilised to determine whether or not a deployed system is able to detect a specific attack and to identify requirements for intrusion detection functionality for the development of new detection methods. Two novel detection methods are presented capable of detecting new and previously unseen executable attack payloads. The detection methods are capable of identifying and enumerating the executable payload’s interactions with the operating system on the targeted host at the time of compromise. The detection methods are further validated using real world data including executable payload attacks.