Thesis / Master of Science (MS)

Malathion-resistance in Drosophila Melanogster was studied in susceptible laboratory strains (CS and OR), a heterogeneous intermediate-resistant strain (C1-39), and a more resistant, artificially selected (with malathion). strain (MH19) by comparing the in vitro metabolism of [¹⁴c]-malathion and [¹⁴c]-malaoxon in crude enzyme preparations made from adult flies. Extracts from all strains were found to contain two enzymatic activities that metabolized malathion and/or malaoxon. One activity degraded malathion to its α- and β-monocarboxylic acids and was designated as malathion-carboxylesterase activity (ME-activity). ME-activity was progressively lost in CS-extracts during reaction with [¹⁴c]-malathion due to inhibition of the enzyme(s) by a tightly bound [¹⁴c]-labeled molecule (not identified) that could not be removed by chromatography on Sephadex G-25. ME-activity, based on initial (0-1 min) rates with or without metyrapone present was similar in all strains and furthermore, the carboxylesterase inhibitors TPP and DEF did not synergize malathion toxicity in either resistant or susceptible strains. It was concluded that carboxylesterase-mediated degradation of malathion was not a factor in the resistance of the C1-39 and MH19 strains. A second enzyme system, microsomal mixed-function oxidases (MFO), converted malathion to malaoxon (activation) and degraded malaoxon to at least two products that were tentatively identified (malaoxon α-and β-monoacids and demethyl-malaoxon). The rate of conversion of malathion to malaoxon was highest in crude extracts of the most resistant MH19 flies, intermediate in C1-39 and could not be detected in the susceptible CS flies while the rate of malaoxon degradation was similar between MH19 and C1-39, but higher than that in the susceptible OR flies. Furthermore, malaoxon (but not malathion) toxicity was most strongly synergized by the MFO-inhibitor MTP in the more resistant strains (MH19 and C1-39) . These results were used along with a previous result that MH19 strain possesses a less sensitive form of the target enzyme, acetylcholinesterase (R.A. Morton, personal communication), to propose a biochemical mechanism that accounts for the increased malathion-resistance of the C1-39 and MH19 strains. / Thesis / Master of Science (MS)

Thesis / Master of Science (MS)

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Background/Aims. Understanding the fundamental mechanisms underlying the development of congenital heart disease and cardiomyopathies is a goal of researchers worldwide, with the ultimate goal being the establishment of effective therapeutics for the amelioration of cardiac dysfunction. Unfortunately these disorders are often polygenic in aetiology, making it difficult for researchers to probe complex interactions that may contribute to the severity of the disease. Over the last decade, the adult fruit fly (Drosophila melanogaster) has emerged as an invaluable tool with which to study the genetic and molecular mechanisms underlying heart function. The aim of my thesis research was to establish the adult fruit fly as a model of human heart function, and to exploit this powerful genetic system to screen for conserved genes affecting the development and function of its cardiac syncytium. Methodology/Results Baseline measures of heart function and other factors contributing to variability in heart function (i.e. age, temperature, and the time of day) were assessed to establish the adult Drosophila heart model. I then performed an a priori RNAi screen, knocking down expression of individual conserved genes via cardiomyocyte-specific overexpression utilising the yeast GAL4/UAS system. Heart-specific ablation of Fermitin 1 and Fermitin 2 (Fit1, Fit2), the two Drosophila orthologs of Kindlin 2 (Kind2, a gene thought to be important for cardiomyocyte-cardiomyocyte junction integrity in human myocardium), caused severe cardiomyopathy characterised by the failure of cardiomyocytes to develop as a functional syncytium and loss of synchrony between cardiomyocytes. I generated a null allele of Fit1 via P-element mobilisation, but this had no impact on heart development or function. Similarly, the silencing of Fit2 failed to affect heart development or function. In contrast, the silencing of Fit2 in the cardiomyocytes of Fit1-null flies disrupted syncytium development, leading to severe cardiomyopathy. Temperature-sensitive cardiac-specific GAL4/GAL80ts lines were also generated, and knockdown of Fit (Fit1 and Fit2) function at different developmental stages was assessed. I observed the strongest effects of Fit knockdown on adult cardiac morphology during stages of heart development and remodelling, with significant cardiomyocyte decoupling. After 3-weeks of Fit knockdown during adulthood, cardiomyocytes were significantly decoupled, and these hearts were significantly arrhythmic compared to control animals. Conclusions/Discussion. My data provide clarity about the role of Kind2 by demonstrating a cell autonomous role for this family in the development of a functional cardiac syncytium in Drosophila. My findings also show that the Fermitins can functionally compensate for each other in order to control syncytium development. Therefore, my thesis demonstrates the power of the fruit fly as a model of human cardiac physiology, and supports the concept that abnormalities in cardiomyocyte KIND2 expression or function may contribute to cardiomyopathies in humans.

Proneural proteins are transcription factors of the bHLH family and have a conserved role in directing neurogenesis from invertebrate to mammals. In Drosophila, proneural proteins are required for early developmental specification of precursor cells of sense organs (SOPs). Despite considerable progress having been made in this field, it remains unknown how proneural proteins organise the well-orchestrated process that facilitates each type of SOP to acquire both generic neuronal properties and individual neuronal subtype identity during the progression from specification to differentiation. To approach this question, we investigate the gene regulatory network by proneural protein Ato by means of the microarray analysis. Ato directs the formation of the Drosophila chordotonal organs (Ch), important proprioceptive sense organs (Jarman et al., 1993b). The microarray study generated a list of candidate Ato target genes (Cachero et al., 2011). My PhD project entails the characterisation of two potential Ato target genes arising from this screen: Rfx and dila. To determine their positions in the gene regulatory network, I analysed the regulation and function of these genes. First, I demonstrated that both Rfx and dila are activated during Ch neurogenesis as direct targets of Ato. This was established by characterising their expression patterns, cis-regulation analyses and identifying the potential Ato binding sites by site-directed mutagenesis. RFX is a well-known ciliogenic regulator (Dubruille et al., 2002; El Zein et al., 2009; Swoboda et al., 2000), and its activation by Ato is consistent with Ch neurons having ciliated dendrites. However, the role of dila was completely unknown, but its sequence suggested that it may be involved in neuronal differentiation rather than gene regulation. I generated several dila mutant alleles and demonstrated that dila mutants exhibit severe uncoordination, due to a series of defects in ciliated neurons. These defects were linked to a disruption in the ciliogenesis machinery, particularly in the process known as intraflagellar transport (IFT). dila mutants also display reduced male fertility because of aberrant basal body function, which leads to a disorder in sperm individualisation. Thus DILA is required for the differentiation of all ciliated cells in Drosophila. Visualisation of tagged protein localised DILA to the basal body and transition zone of the sensory cilia. Further analysis revealed the genetic interaction between DILA and UNC (another basal body protein) during ciliogenesis. Taken together I propose that DILA regulates IFT at the base of the cilia in collaboration with UNC. Given that dila is an evolutionarily conserved gene, dila homologues could be candidate genes for human ciliopathies. Rfx is essential for ciliogenesis in both Ch and the external sense (ES) organs, which have distinctive cilia. Despite of this common role of RFX, I discovered that Rfx is expressed differently in Ch and ES lineages, which led me to hypothesise that the difference in Rfx expression modulates ciliogenesis in these two lineages. I obtained preliminary data that support this hypothesis. Overall, my study demonstrates important links between Ato and the regulation of ciliogenesis, which is an important process in Ch neuron differentiation. The data support a model in which Ato controls ciliogenesis both directly (e.g. via activating a ciliary genes like dila) and indirectly (e.g. via regulating the transcriptional factors essential for ciliogenesis, like RFX).

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