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In D. melanogaster, resistance to DDT is conferred by the upregulation of a cytochrome P450 enzyme, CYP6G1. Resistant flies have tandemly duplicated Cyp6g1 alleles that possess the LTR (Long Terminal Repeat) of an Accord retrotransposon inserted in the cis-regulatory region, 291bp upstream of the transcription start site. This DDT resistance allele (DDT-R) has been shown to have pleiotropic fitness benefits for female flies in at least one genetic background and with evidence of sexually antagonistic selection at this locus. In this thesis, I first review the role of transposable elements in conferring insecticide resistance and the evidence to date regarding the pleiotropic effects of DDT-R in D. melanogaster. By conducting life history and behavioural tests on flies of two genetic backgrounds I examine the sex-specific effects of expressing DDT-R in the absence of DDT. Finally I develop a single locus population genetics model based on these sex-specific effects and test the model using replicate laboratory populations. The first main finding is that DDT-R incurred a male mating cost that depended on the genetic background in which DDT-R was found and that this cost coincided with strong epistasis between genetic background and DDT-R that influenced male size (Chapter 3). Following on from this result, it was confirmed that the effect of DDT-R on male size does contribute to lowered mating success but does not fully explain this fitness cost (Chapter4). Additionally, resistant males were found to have a lowered rate of courtship behaviour driven by aborted chasing of females and lower male-male aggression than susceptible males (Chapter 4). Fitness assays in wild caught strain females revealed that DDT-R confers a fecundity increase but unlike previous work, no offspring viability increases were detected (Chapter 5). Thus as with male costs, specific pleiotropic female fitness benefits to resistance depend on genetic background. Modelling of DDT-R using a simple single-locus approach (Chapter 6) provides, for the first time, a unifying explanation for past and present DDT-R frequencies in nature and in old laboratory populations. The model is consistent with an old origin for the original DDT-R mutation held at low equilibrium frequency through balancing selection of a sexually antagonistic nature. It is also consistent with continued near fixation of DDT-R long after discontinued use and matches empirical observations in laboratory populations of the Canton-S background.

Two different kinds of stabilising homeostatic behaviour have been observed in neurons. The first type involves the cell-autonomous maintenance of a cell-identity-based level of electrical activity. Neurons continually monitor their own electrical activity and can adjust many intracellular parameters, such as membrane ion channel densities, to keep this activity within a tight physiological range. The second type of homeostatic behaviour shares the same goal, to maintain a fixed level of electrical activity, but instead of adjusting intracellular parameters, the neuron recruits its synaptic partners to assist in maintaining a genetically prescribed activity level. This behaviour is most easily observed when a neuron is either electrically silenced by expressing an inwardly-rectifying potassium channel or rendered less sensitive to neurotransmitter through mutation of its postsynaptic receptors. Both of these perturbations result in increased synaptic drive from the presynaptic cells, either through increasing the number of neurotransmitter release sites or increasing the probability of release from single release sites. Many genes that are instrumental in the second type of homeostatic behaviour have been identified, mainly at the neuromuscular junction in the peripheral nervous system. However, studies on transsynaptic homeostatic compensation in an intact central nervous system have been few and far between. Also, which, if any, of the homeostatic genes are transcriptionally regulated in the nucleus after the onset of transsynaptic homeostatic adjustment, has not been adequately addressed. This thesis has developed a system to measure transcriptome-wide gene expression levels in presynaptic circuit elements after altering the firing properties of the downstream circuit in the CNS. Many transcriptionally regulated genes have been identified and are now being tested for their potential use as reporters for transsynaptic transcriptional regulation. It might be possible to capitalise on endogenous homeostatic signalling pathways to gain genetic access to synaptically connected neurons.

Senescence is the age-related progressive deterioration of physiological processes leading to an increased likelihood of death and is a phenomenon that occurs nearly universally throughout all the world’s organisms. This thesis initially investigated the impact of ageing on the adult Drosophila melanogaster cellular immune response and demonstrated that the cellular immune response in D. melanogaster adults did experience an age-dependent decline in function. There was a striking reduction in haemocyte ability to phagocytose foreign particles with up to 30% less phagocytosis occurring in four week old flies compared to one week olds. Haemocyte number also declined in female flies by up to 32% across these ages. An exploration into the mechanisms that could underlie these observed senescent declines in haemocyte number and function revealed that the age-dependent reduction in the circulating haemocyte population occurred regardless of whether flies were unharmed, wounded or infected. The loss of phagocytosis ability in haemocytes in ageing flies was shown to be a cell autonomous process; there was an equal age-dependent decline (~13%) in haemocyte phagocytic activity in both in vivo and ex vivo assays. However, an attempt to identify phagocytic receptor systems that drove senescence in haemocyte function was unsuccessful. The contribution of the cellular immune response in determining survival following a fungal infection was not conclusively demonstrated, however flies with reduced Dif expression had significantly increased pathogen susceptibility. Although pathogen resistance can decline due to immune senescence, disease defence may also be enhanced as an animal’s life progresses through the formation of immunological memories of prior microbial encounters. This thesis revealed that the cellular immune response in D. melanogaster provides a strong, broadly specific and relatively long-lasting immunological priming response. Haemocytes phagocytosed up to 33% more microbes per cell during a secondary encounter, and up to 50% more if flies had received two homologous primes. This was not general immune upregulation as a heterologous microbial encounter caused a reduction in the phagocytic ability of haemocytes compared to controls. The level of enhancement in the phagocytic ability of haemocytes also declined with the age of the fly, meaning that the ability to develop a primed response senesced. These results are unprecedented in Drosophila and challenge our conventional interpretation of immune senescence because individual immune history has been shown to shape later cellular immune responses. Ageing is a complex and variable process. Some of the differences observed in ageing rates between populations can be due to different selection pressures. Natural selection acts on genetic variation within a population to increase fitness whereas host-parasite interactions predominantly influence genes related to immune parameters. Many genes have pleiotropic effects as well as there being potential trade-offs between investment in longevity, reproduction and immunity. To explore potential genetic variation in immune and life history traits and whether variation in immune parameters negatively influenced other life history traits related to ageing, a panel of outcrossed genotypes of D. melanogaster were assessed. As the flies were derived from individuals originally sourced from a natural population, the results suggest that a striking amount of genetic variation in immune and life history traits is present in wild populations. However no significant correlations between genetic variation in ageing and genetic variation in investment in immunity were identified. Though, perhaps not surprisingly, no key biomarker of ageing in D. melanogaster was identified; this thesis has contributed some significant findings on the effects of ageing on adult D. melanogaster especially relating to their cellular immune response.

The goal of my doctoral work is to understand how proteins involved in vesicle trafficking contribute to proper animal development. To understand aspects of this process, I studied how two vesicle trafficking proteins, Liquid facets(Lqf)/epsin1 and D-Epsin-Related, affect Drosophila eye development. I determined that Lqf, an endocytosis protein, together with Fat facets (Faf), a deubiquitinating enzyme, regulate the Notch and Delta signaling in the developing Drosophila eye. Notch signaling pathway is used in most developmental processes and is dependent on its ligand Delta. Faf deubiquitinates Lqf in the signaling cells, thereby increasing Lqf protein levels and also levels of Delta endocytosis. This event is necessary for Notch activation in neighboring cells. Lqf probably works in concert with the E3 ubiquitin ligase Neuralized (Neur), which ubiquitinates Delta. These conclusions are consistent with a relatively new model describing an obligate role for endocytosis in the signaling cells to effect activation in neighboring cells. To understand how Lqf functions mechanistically in this process, I performed a structure/function analysis of the Lqf protein. Lqf proteins with strategic deletions of certain functional domains were tested for their ability to function in vivo. The major result of these experiments is that the N-terminal ENTH domain of Lqf and a protein without the ENTH domain each retain significant activity. This suggests that Lqf has two functions: the ENTH domain function and the ENTH-less function. These data are in contrast with the most popular model suggesting that ENTH-less epsins are non-functional proteins. I present possible models for how ENTH-less epsins may retain function. The final part of my thesis focuses on D-Epsin-Related (D-Epsin-R) protein. I showed that D-Epsin-R is a Golgi protein, like its homologs. Surprisingly, D-Epsin-R ENTH domain is not required for function because an ENTH-less D-Epsin-R can substitute for endogenous D-Epsin-R. Also, D-Epsin-R has essential and probably specific developmental roles in the eye as D-Epsin-R mutants exhibit impaired cell growth. This work suggests that epsins are specific components of certain developmental pathways. / text

<p>Calcineurin is both necessary and sufficient to induce cardiac hypertrophy, an independent risk factor for arrhythmia, dilated cardiomyopathy, heart failure, and sudden cardiac death. However, current knowledge of the downstream effectors of calcineurin is limited. My study utilizes <italic>Drosophila melanogaster</italic> to 1) establish a reliable model for discovering novel modifiers of calcineurin-induced cardiomyopathy; and 2) discover and characterize novel modifiers of calcineurin-induced cardiomyopathy.</p><p>In this study, I generated sensitized <italic>Drosophila</italic> lines expressing constitutively active calcineurin (CanA<super>act</super>) that was either fused to yellow fluorescent protein (YFP) or a Flag epitope (Flag-tagged) specifically in the heart using the cardiac-specific tinC driver (<italic>tinC-CanA<super>act</super></italic>). These sensitized lines displayed significant cardiac enlargement as assayed via optical coherence tomography (OCT), histology, and confocal microscopy. The feasibility of this method was established by testing <italic>Drosophila</italic> expressing deficiency of a known calcineurin modifier, Mef2. </p><p>Employing a targeted deficiency screen informed by calcineurin modifier screens in the eye and mesoderm, Galactokinase (<italic>Galk</italic>) was discovered as a novel modifier of calcineurin-induced cardiomyopathy in the fly through 1) genetic deficiencies, transposable elements, and RNAi disrupting <italic>Galk</italic> expression rescued <italic>tinC-CanA<super>act</super></italic>-induced cardiomyopathy; and 2) transposable element in <italic>Galk</italic> rescued <italic>tinC-CanA<super>act</super></italic>-induced decreased life span. Further characterization identified that the genetic disruption of <italic>Galk</italic> rescued CanA<super>act</super>-induced phenotypes driven in the posterior wing, but not ectodermaly, mesodermaly, or ubiquitously driven phenotypes. In a separate region, genetic disruption of the galactoside-binding lectin, galectin, was also found to rescue <italic>tinC-CanA<super>act</super></italic>-induced cardiac enlargement.</p><p>Together, these results characterize <italic>tinC-CanA<super>act</super></italic>-induced cardiac enlargement in the fly, establish that the <italic>tinC-CanA<super>act</super></italic> sensitized line is a reliable model for discovering novel calcineurin regulators and suggest that galactokinase and galectin-regulated glycosylation is important for calcineurin-induced cardiomyopathy. These results have the potential to provide insight into new treatments for cardiac hypertrophy.</p> / Dissertation

L’objectif général de ma thèse concerne l’étude de la régulation de l’activité cardiaque chez Drosophila melanogaster. Différentes questions ont été abordées : i) la régulation par le pH de l’activité cardiaque à travers l’étude d’un Transporteur du Bicarbonate dépendant du Na+, NDAE1 ; ii) l’implication du canal TRP Painless dans la mécanosensibilité du cœur ; iii) l’élaboration de tests quantitatifs permettant de mesurer le vieillissement cardiaque chez la mouche adulte. Le Na+-Driven Anion Exchanger (NDAE1) constitue l’unique transporteur chez la Drosophile capable de transporter le Bicarbonate dépendant du Na+, alors que l’on trouve dans le génome des mammifères 7 de ces transporteurs appartenant à la famille SLC4. NDAE1 permet l’échange de protons et de Cl- avec le Na+ et HCO3- et agit de manière réversible. Etant donné l’importance potentielle et reconnue de ce type d’échangeur durant certaines pathologies cardiaques intervenant par exemple lors d’épisodes d’ischémie-reperfusion, j’ai analysé sa fonction dans l’activité cardiaque. De manière surprenante, l’inactivation du gène spécifiquement dans le tube cardiaque par interférence à l’ARN n’a aucun effet sur les paramètres mesurables de l’activité cardiaque dans des conditions basales d’élevage, ni sur la viabilité. En revanche, la fonction de NDAE1 peut être révélée dans des conditions de stress où l’on déséquilibre l’homéostasie des ions transportées dans l’échange dépendant de nDAE1. Ainsi, une acidose provoquée dans les individus privés de la fonction de NDAE1 génère de très fortes arythmies, qui sont moins présentes dans les animaux de type sauvage, et conduisent à des arrêts cardiaques définitifs. En outre, arythmies et arrêts cardiaques sont irréversibles quand le pH physiologique est restoré, contrairement aux contrôles qui retrouvent complètement leur activité cardiaque normale. De même l’activité de NDAE1 est requise pour mieux résister aux stress provoqués par l’absence de Na+, de Cl- et de HCO3- dans le milieu extracellulaire et d’adapter à un choc osmotique. En outre, j’ai mis en évidence une forte interaction génétique de ndae1 avec ncx, qui code pour l’échangeur Sodium-Calcium, et dont la fonction est de réguler l’homéostasie calcique et sodique. Cette étude constitue la première démonstration in vivo de la fonction cardiaque des Transporteurs du Bicarbonate dépendant du Na+. J’ai d’autre part contribué à l’étude de la réponse des cardiomyocytes aux stress mécaniques et participé à la démonstration que Painless, un canal TRPA de la Drosophile, était requis pour cette réponse. Finalement, dans le cadre du programme « Identification of genetic markers of cardiac aging in Drosophila », j’ai cherché à proposer des tests capables de mesurer le déclin des performances cardiaques avec l’âge. Parmi ceux-ci, le plus prometteur consiste en une quantification des arythmies mesurée par l’analyse in vivo détaillée des battements cardiaques / Mechanotransduction modulates cellular functions as diverse as migration, proliferation, differentiation and apoptosis. It is crucial for organ development and homeostasis and leads to pathologies when defective. However, despite considerable efforts made in the past, the molecular basis of mechanotransduction remains poorly understood.Here, we have investigated the genetic basis of mechanotransduction in Drosophila. We show that the fly heart senses and responds to mechanical forces by regulating cardiac activity. In particular, pauses in heart activity are observed under acute mechanical constraints in vivo. We further confirm by a variety of in situ tests that these cardiac arrests constitute the biological force-induced response.In order to identify molecular components of the mechanotransduction pathway, we carried out a genetic screen, based on the dependence of cardiac activity upon mechanical constraints and identified Painless, a TRPA channel. We observe a clear absence of in vivo cardiac arrest following inactivation of painless and further demonstrate that painless is autonomously required in the heart to mediate the response to mechanical stress. Furthermore, direct activation of Painless is sufficient to produce pauses in heartbeat, mimicking the pressure-induced response. Painless thus constitutes part of a mechanosensitive pathway that adjusts cardiac muscle activity to mechanical constraints.This constitutes the first in vivo demonstration that a TRPA channel can mediate cardiac mechanotransduction. Furthermore, by establishing a high-throughput system to identify the molecular players involved in mechanotransduction in the cardiovascular system our study paves the way for understanding the mechanisms underlying a mechanotransduction pathway.

Stress can be defined as factors which reduce an individual’s survival and/or reproductive ability. Certain stressors strike harder against individuals the more harmful mutations they carry, thus increasing selection against harmful mutations. The aim of this project is to test if exposure to stress, during many generations, decreases the frequency of harmful mutations and lead to more adapted individuals, or if specific adaptations to the stressor override this effect and results in reduced adaptation in the original environment through trade-offs. To test these hypotheses, I use selection lines of Drosophila melanogaster, where the flies during the larval state either have been exposed to media with reduced nutritional value or a heat shock treatment over 22 generations. The results from this experiment show that the selection lines have adapted to their novel environments, since survival of the larvae had increased in the novel environment they had been exposed to for 22 generations. However, survival of selection lines were if anything decreased rather than elevated in the original environment. A plausible explanation to these results is that adaptations to stressors occur both through trade-offs and reduced frequency of generally harmful mutations, but that the effect of the former possibly is larger than the latter. / Stress kan definieras som faktorer som minskar en individs överlevnad och/eller reproduktiva förmåga. Vissa stressmiljöer slår relativt hårdare mot individer ju fler skadliga mutationer de bär, och ökar därför selektionstrycket mot skadliga mutationer. Detta projekt har som mål att testa om exponering mot sådana stressmiljöer, under flera generationer, minskar frekvensen av skadliga mutationer och leder till generellt bättre anpassade individer, eller om specifika anpassningar till stressmiljön överskuggar denna effekt och via trade-offs leder till individer som är sämre anpassade till ursprungsmiljön. För att testa dessa hypoteser använder jag mig här av selektionslinjer av Drosophila melanogaster, där flugorna under larvstadiet antingen utsatts för en näringsfattig miljö eller en värmechock under 22 generationer. Resultaten från detta experiment visar en tydlig evolutionär respons, i och med att larvöverlevnaden ökat för de selekterade linjerna i den stressmiljö de utsatts för efter 22 generationer. Test av överlevnad i ursprungsmiljön visar dock ingen signifikant skillnad mot kontrollinjerna, men om något att de selekterade linjerna klarade sig något sämre. Dessa resultat tyder om något på att anpassningar som skett till den nya miljön på bekostnad av anpassningar i ursprungsmiljön (via trade-offs) överskuggar ökad anpassning via en minskad frekvens av generellt skadliga mutationer.

Traditional studies of simple perceptual choice tasks in vertebrates identified behavioural characteristics of deliberate decision-making that guided the development of general mathematical models, and the search for neurophysiological correlates. Current experimental and modelling efforts aim to uncover biophysical and circuit level mechanisms of decision-making processes. However, genetic manipulability constraints and lack of high-throughput assays make further progress in vertebrate studies a steep endeavour. In this thesis I studied decision-making in Drosophila melanogaster in trained two-alternative forced-choice olfactory tasks with varying stimulus contrast, using a high-resolution single fly behavioural assay. Analysing accuracy and reaction time as a function of task difficulty (i.e., stimulus contrast) showed that flies display behavioural characteristics of evidence accumulation processes, a signature of vertebrate decision-making: reaction times increased and perceptual accuracy declined as stimulus contrast decreased. Mutants for the gene encoding the transcription factor FoxP took longer than wild-type flies to form decisions of similar or reduced accuracy, especially in difficult tasks. Using the putative FoxP promoter to ascertain FoxP expression identified subsets of mushroom body intrinsic Kenyon cells, in &alpha;&beta; core and &gamma; neurons, as potential sites of FoxP action. Disrupting FoxP expression or decreasing neuronal excitability specifically in &alpha;&beta; core neurons mimicked the phenotype observed in FoxP mutants. FoxP expression therefore affects the development or function of &alpha;&beta; core neurons in the progression of a decision process towards commitment. To identify molecular processes involved in evidence integration regulated by FoxP I further screened 2nd and 3rd chromosome deficiency lines in a sensitised FoxP mutant background, uncovering genomic regions of interest for further study. Finally, analysing naive performance in tasks of increasing difficulty showed that naive discriminations are faster and less accurate than trained ones, pointing to the existence of two decision-making systems. FoxP mutants appear to engage the slower, more accurate decision making system and the mushroom body seems to be involved in naive discriminations. The molecular and neuronal players involved in olfactory decision making in Drosophila melanogaster uncovered in this thesis will allow researching decision making systems to an unprecedented level of detail.

Drosophila melanogaster has been extensively used as a model organism to study diverse facets of biology, including host-pathogen interactions and the basic biology of its pathogens. I have used the fruit fly as a model to study elementary aspects of Nora virus biology, such as the role of the different proteins encoded by the virus genome. Nora virus, an enteric virus transmitted via the feca-oral route, does not cause any obvious pathology in the fly, although the infection is persistent. Nora virus genome consists of a positive strand RNA that is translated in four open reading frames (ORF).  Since sequence homology studies did not yield much information about the different Nora virus proteins, I have used the cDNA clone of the virus to construct mutants to identify the specific function of each protein. My results have shown that, 1) The protein(s) encoded by ORF 1 are crucial for the replication of the virus genome. 2) The C-terminus of the ORF 1-encoded protein (VP1), is an inhibitor to the RNAi pathway. 3) The transmembrane domain in the N-terminus of the ORF2-encoded protein (VP2) is important for the formation of Nora virus virions. 4) The ORF 3-encoded protein (VP3) forms α-helical trimers and this protein is essential for the stability of Nora virus capsid.                                                     I have also performed RNA sequencing to investigate the transcriptional response of D. melanogaster in response to Nora virus infection and my results indicate that,                        5) The upregulation of genes related to cellular stress and protein synthesis and the downregulation of basal digestive machinery, together with the induction of upd3, implies major gut epithelium damage and subsequent regeneration.