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.

Triumphs and tribulations in the psychotherapy of schizophrenia : reflections on a pilot study of metacognitive narrative psychotherapy

Schizophrenia results in a profound disruption of one’s capacity to make sense of mental states, coherently narrate self-experiences, and meaningfully relate to others. While current treatment options for people with schizophrenia tend to be symptom-focused, experience in designing and implementing a study focusing on enhancing sense of self demonstrates the feasibility of developing and implementing models of treatment that prioritize the subjective distress and self-experience of people with schizophrenia. There is emerging research evidence, based upon dialogical theory of self, that posits the potential of people with deficits of self to engage in meaningful therapeutic relationships and work toward greater integrity of self and degrees of recovery. The challenge is to translate these ideas into a research methodology that can be successfully applied within therapeutic contexts with people who meet the diagnostic criteria for schizophrenia. Based upon dialogical theory, we developed a principle-based manual for metacognitive narrative psychotherapy: a psychological approach to the treatment of people with schizophrenia, which aims to enhance metacognitive capacity and ability to narrate self-experiences. Five phases of treatment were identified: (1) developing a therapeutic relationship, (2) eliciting narratives, (3) enhancing metacognitive capacity, (4) enriching narratives, and (5) living enriched stories. Proscribed practices were also identified. We then implemented the manual within a university clinic context. Six therapists were trained to implement the model and, in turn, provided therapy to 11 patients who completed 12 to 24 months of treatment. Participants were assessed on metacognitive capacity, narrative coherence, narrative richness, self-reported recovery, and symptomatology at three points in time over the course of therapy. Contrary to expectations, participants were highly engaged in the therapeutic process, with minimal dropout. Overall, over 75% of participants evidenced improvement in their level of recovery over the course of therapy. The manualization and outcome findings demonstrate the feasibility of applying such interventions to a broader clinical population.

Health consequences of exposure to e-waste : a systematic review

Background The population exposed to potentially hazardous substances through inappropriate and unsafe management practices related to disposal and recycling of end-of-life electrical and electronic equipment, collectively known as e-waste, is increasing. We aimed to summarise the evidence for the association between such exposures and adverse health outcomes. Methods We systematically searched five electronic databases (PubMed, Embase, Web of Science, PsycNET, and CINAHL) for studies assessing the association between exposure to e-waste and outcomes related to mental health and neurodevelopment, physical health, education, and violence and criminal behaviour, from Jan 1, 1965, to Dec 17, 2012, and yielded 2274 records. Of the 165 full-text articles assessed for eligibility, we excluded a further 142, resulting in the inclusion of 23 published epidemiological studies that met the predetermined criteria. All studies were from southeast China. We assessed evidence of a causal association between exposure to e-waste and health outcomes within the Bradford Hill framework. Findings We recorded plausible outcomes associated with exposure to e-waste including change in thyroid function, changes in cellular expression and function, adverse neonatal outcomes, changes in temperament and behaviour, and decreased lung function. Boys aged 8–9 years living in an e-waste recycling town had a lower forced vital capacity than did those living in a control town. Significant negative correlations between blood chromium concentrations and forced vital capacity in children aged 11 and 13 years were also reported. Findings from most studies showed increases in spontaneous abortions, stillbirths, and premature births, and reduced birthweights and birth lengths associated with exposure to e-waste. People living in e-waste recycling towns or working in e-waste recycling had evidence of greater DNA damage than did those living in control towns. Studies of the effects of exposure to e-waste on thyroid function were not consistent. One study related exposure to e-waste and waste electrical and electronic equipment to educational outcomes. Interpretation Although data suggest that exposure to e-waste is harmful to health, more well designed epidemiological investigations in vulnerable populations, especially pregnant women and children, are needed to confirm these associations. Funding Children's Health and Environment Program, Queensland Children's Medical Research Institute, The University of Queensland, Australia.

Environmental exposures : an underrecognized contribution to noncommunicable diseases

Previous attempts to determine the degree to which exposure to environmental factors contribute to noncommunicable diseases (NCDs) have been very conservative and have significantly underestimated the actual contribution of the environment for at least two reasons. Firstly, most previous reports have excluded the contribution of lifestyle behavioral risk factors, but these usually involve significant exposure to environmental chemicals that increase risk of disease. Secondly, early life exposure to chemical contaminants is now clearly associated with an elevated risk of several diseases later in life, but these connections are often difficult to discern. This is especially true for asthma and neurodevelopmental conditions, but there is also a major contribution to the development of obesity and chronic diseases. Most cancers are caused by environmental exposures in genetically susceptible individuals. In addition, new information shows significant associations between cardiovascular diseases and diabetes and exposure to environmental chemicals present in air, food, and water. These relationships likely reflect the combination of epigenetic effects and gene induction. Environmental factors contribute significantly more to NCDs than previous reports have suggested. Prevention needs to shift focus from individual responsibility to societal responsibility and an understanding that effective prevention of NCDs ultimately relies on improved environmental management to reduce exposure to modifiable risks.