Two problems hampering efforts to produce salt-tolerant plants through constitutive expression of transgenes include: 1. Spatial control. Particular cell-types must respond specifically to salt stress to minimise the amount of Na⁺ delivered to the shoot; and, 2. Temporal control. Transgenes are typically expressed in plants at similar levels through time, irrespective of the stress encountered by the plant, which may exacerbate pleiotropic effects and means that, particularly in low-stress conditions, costly and/or detrimental metabolic processes may be active, thus reducing yield. To address these issues, Gateway® destination vector constructs were developed combining the GAL4 UAS (upstream activating sequence) with the ethanol-inducible gene expression system to drive inducible cell-specific expression of Na⁺ transporter transgenes (or to silence salt transporter transgenes inducibly and cell-specifically). Rice (Oryza sativa L. cv. Nipponbare) GAL4-GFP enhancer trap lines (Johnson et al., 2005: Plant J. 41, 779-789) that express GAL4 and GFP specifically in either the root epidermis or xylem parenchyma (and therefore ‘trap’ cell-type specific enhancer elements) were transformed with this GAL4 UAS – ethanol switch construct, thereby allowing both spatial and temporal control of transgenes. In preliminary experiments, the expression system successfully limited the expression of RFP to specific cell-types after induction with ethanol. Other genes expressed using this system include PpENA1, a Na⁺-extruding ATPase from the moss, Physcomitrella patens, and AtHKT1;1, a Na ⁺ transporter from Arabidopsis thaliana. The two enhancer trap rice lines were also transformed with the GAL4 UAS driving stable expression of AtHKT1;1 and PpENA1 specifically in root epidermal or xylem parenchyma cells. Expression of AtHKT1;1 in root epidermal cells reduced Na⁺ accumulation in the shoots, while expression in the root xylem parenchyma appeared to have little effect on shoot Na⁺ accumulation. Using cryo-scanning electron microscopy (SEM) X-ray microanalysis, the outer cells of the roots of the line expressing AtHKT1;1 in the epidermal cells were found to accumulate higher levels of Na⁺ than the parental enhancer trap line. Additionally, this line had decreased unidirectional ²²Na⁺ influx. Similar results were observed for plants expressing AtHKT1;1 driven by the CaMV 35S / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1325289 / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2008

Two problems hampering efforts to produce salt-tolerant plants through constitutive expression of transgenes include: 1. Spatial control. Particular cell-types must respond specifically to salt stress to minimise the amount of Na⁺ delivered to the shoot; and, 2. Temporal control. Transgenes are typically expressed in plants at similar levels through time, irrespective of the stress encountered by the plant, which may exacerbate pleiotropic effects and means that, particularly in low-stress conditions, costly and/or detrimental metabolic processes may be active, thus reducing yield. To address these issues, Gateway® destination vector constructs were developed combining the GAL4 UAS (upstream activating sequence) with the ethanol-inducible gene expression system to drive inducible cell-specific expression of Na⁺ transporter transgenes (or to silence salt transporter transgenes inducibly and cell-specifically). Rice (Oryza sativa L. cv. Nipponbare) GAL4-GFP enhancer trap lines (Johnson et al., 2005: Plant J. 41, 779-789) that express GAL4 and GFP specifically in either the root epidermis or xylem parenchyma (and therefore ‘trap’ cell-type specific enhancer elements) were transformed with this GAL4 UAS – ethanol switch construct, thereby allowing both spatial and temporal control of transgenes. In preliminary experiments, the expression system successfully limited the expression of RFP to specific cell-types after induction with ethanol. Other genes expressed using this system include PpENA1, a Na⁺-extruding ATPase from the moss, Physcomitrella patens, and AtHKT1;1, a Na ⁺ transporter from Arabidopsis thaliana. The two enhancer trap rice lines were also transformed with the GAL4 UAS driving stable expression of AtHKT1;1 and PpENA1 specifically in root epidermal or xylem parenchyma cells. Expression of AtHKT1;1 in root epidermal cells reduced Na⁺ accumulation in the shoots, while expression in the root xylem parenchyma appeared to have little effect on shoot Na⁺ accumulation. Using cryo-scanning electron microscopy (SEM) X-ray microanalysis, the outer cells of the roots of the line expressing AtHKT1;1 in the epidermal cells were found to accumulate higher levels of Na⁺ than the parental enhancer trap line. Additionally, this line had decreased unidirectional ²²Na⁺ influx. Similar results were observed for plants expressing AtHKT1;1 driven by the CaMV 35S / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1325289 / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2008

Muscle contraction is a highly fine-tuned process that requires the precise and timely construction of large protein sub-assemblies to form sarcomeres, the individual contractile units. Mutations in many of the genes encoding constituent proteins of this macromolecular machine result in defective functioning of the muscle tissue, and in humans, often lead to myopathic conditions like cardiomyopathies and muscular dystrophies, which affect a considerable number of people the world over. As more information regarding causative mutations becomes available, it becomes imperative to explore mechanisms of muscle development, maintenance and pathology. In striated muscles, contraction is regulated by the thin filament-specific tropomyosin (Tm) – troponin (Tn) complex (Ca2+-binding troponin-C, inhibitory troponin-I and tropomyosin-binding troponin-T). These troponin subunits are present in 1:1:1 ratio on thin filaments, with 1 Tm-Tn complex present on every 7th actin molecule. This stoichiometry is tightly regulated, and disturbances have been associated with functional defects. Each of these proteins has multiple isoforms, whose expression is controlled both spatially and temporally. The expression of specific combination of isoforms confers specific contractile properties to each muscle subtype. Drosophila melanogaster has been a preferred model of choice to study various aspects of muscle development for decades. In this study, the Indirect Flight Muscles (IFMs) of Drosophila have been used to investigate developmental and functional roles of two temporally regulated isoforms of a vital structural and regulatory component of the sarcomere – Troponin T (TnT). On a larger scale, whole genome expression profiles of mutants that are null for major myofbrillar proteins have also been discussed. IFMs serve as an excellent model system to address these questions, owing to the extreme ease of genetic manipulability in this system, and high degree of homology between mammalian and Dipteran cytoskeletal proteins. Chapter 1 covers basics of muscle biology, and the role of TnT in muscle contraction. Phenomena responsible for generating diversity in genes encoding muscle proteins – alternative splicing and isoform switching – have also been discussed. These mechanisms are highly conserved, as are patterns of TnT splicing and isoform expression across phyla. Mutations leading to altered splicing patterns lead to myopathic conditions, and the importance of model systems to study muscle biology has been emphasized. The advantages of studying Drosophila IFMs and a comprehensive overview of IFM development has been covered. The resources and experimental tools used have been described in Chapter 2. Two isoforms of TnT are alternatively spliced in the Drosophila thorax – one containing alternative exon 10a (expressed in adult IFMs and jump muscle); and one containing alternative exon 10b (expressed in pupae and newly eclosed flies). These exons are spliced in a mutually exclusive manner, and defects in splicing have been reported to cause uncontrolled, auto-destructive contractions. In Chapter 3, a splice mutant of TnT, up1, has been discussed, with respect to its developmental profile. Transgenic rescue experiments with two separate isoforms demonstrate rescue at the structural as well as functional level. Transgenic over-expression, however, leads to functional abnormalities, highlighting the importance of stoichiometry in multi-protein complexes. In Chapter 4, molecular signals that bring about the developmentally regulated TnT isoform switch are discussed. A splicing factor, Muscleblind, has been transgenically knocked down in normal and mutant IFMs to study effects on muscle function. Chapter 5 looks at whole genome transcriptional alterations in muscles null for either actin or myosin. All significant expression changes have been classified into categories based on different biological processes, and an attempt to differentiate generic muscle responses from filament-specific responses has been made. In conclusion, the studies have highlighted the importance of TnT isoform switching, and that extended expression of a pupal stage-specific isoform can partially compensate for loss of the adult isoform. Also, in the absence of major myofibrillar proteins, stress response pathways like heat shock response and protein degradation pathways are activated, along with a subset of metabolic responses that are unique to the thin or thick filament systems.

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Proper neuronal function requires the preservation of appropriate neural excitability. An adaptive increase in neural excitability after exposure to agents that depress neuronal signaling blunts the sedative drug effects upon subsequent drug exposure. This adaptive response to drug exposure leads to changes in drug induced behaviors such as tolerance, withdrawal and addiction. Here I use Drosophila melanogaster to study the cellular and neuronal components which mediate behavioral tolerance to the anesthetic benzyl alcohol. I demonstrate that rapid tolerance to benzyl alcohol is a pharmacodynamic mechanism independent of drug metabolism. Furthermore, tolerance is a cell autonomous response which occurs in the absence of neural signaling. Using genetic and pharmacological manipulations I find the synapse to play an important role in the development of tolerance. In addition, the neural circuits that regulate arousal and sleep also alter benzyl alcohol sensitivity. Beyond previously described transcriptional mechanisms I find a post-translational role of the Ca2+-activated K+-channel, slowpoke in the development of tolerance. Finally, I explore a form of juvenile onset tolerance, which may have origins that differ from rapid tolerance. The implications of this study go beyond tolerance in Drosophila melanogaster to benzyl alcohol and can shed light on human drug tolerance, withdrawal and addiction. / text