The steroid hormone ecdysone plays vital roles during Drosophila development. Pulses of 20E during Drosophila life cycle function as temporal cues, signaling the onset of metamorphic processes, including the stage specific programmed cell death of larval tissues. Ecdysone is the critical developmental cue orchestrating the metamorphic reformation of CNS, resulting in the formation of adult-specific neural circuitry. Ecdysone signaling is transduced by a heterodimeric receptor complex formed between two nuclear receptors: EcR and Ultraspiracle (USP). There are 18 nuclear receptors known in Drosophila and EcR is the only receptor whose functions in neuronal PCD have been well recognized. Therefore, the current study is aimed to define the role of nuclear receptors in neuronal cell death mechanisms in Drosophila. Here, I examine the function of nuclear receptors in PCD of two groups of peptidergic neurons: vCrz and CCAP. EcR and USP receptor complex on activation results in the coordinated transcriptional regulation of a host of transcription factors regulating genes essential for PCD. USP plays a dual role in ecdysone response, as its function is necessary for both activation and repression of ecdysone primary response genes. I have developed a possible dominant-negative mutant USP (usp3), and expressed it in flies using the GAL4-UAS system to illustrate the role of USP in ecdysone mediated PCD of vCrz neurons. Targeted expression of usp3 in corazonin neurons results in a complete blockage of PCD pathway. Another interacting partner of USP, Drosophila Hormone Receptor 38, however shows no involvement in PCD of vCrz neurons. I have also designed an ecdysone sensor to monitor the developmental timing of EcR activation in vCrz neurons. Further, I investigate the survival factors required for preventing the untimely PCD of these two groups of neurons. The study reveals that DIAP1 is required for the survival of larval vCrz and CCAP neurons. Also, the nuclear receptor E75 is shown to be critical for preventing premature PCD of CCAP neurons.

The vacuolar H+-ATPase (V-ATPase) in the apical membrane of blowfly (Calliphora vicina) salivary gland cells energizes the secretion of a KCl-rich saliva in response to the neurohormone serotonin (5-HT). We have shown previously that exposure to 5-HT induces a cAMP-mediated reversible assembly of V-0 and V-1 subcomplexes to V-ATPase holoenzymes and increases V-ATPase-driven proton transport. Here, we analyze whether the effect of cAMP on V-ATPase is mediated by protein kinase A (PKA) or exchange protein directly activated by cAMP (Epac), the cAMP target proteins that are present within the salivary glands. Immunofluorescence microscopy shows that PKA activators, but not Epac activators, induce the translocation of V1 components from the cytoplasm to the apical membrane, indicative of an assembly of V-ATPase holoenzymes. Measurements of transepithelial voltage changes and microfluorometric pH measurements at the luminal surface of cells in isolated glands demonstrate further that PKA-activating cAMP analogs increase cation transport to the gland lumen and induce a V-ATPase-dependent luminal acidification, whereas activators of Epac do not. Inhibitors of PKA block the 5-HT-induced V-1 translocation to the apical membrane and the increase in proton transport. We conclude that cAMP exerts its effects on V-ATPase via PKA.

Alzheimer’s disease is a common and very costly disease in today’s society. The hallmarks of the disease are the formation of two proteinaggregates, amyloid plaques containing Aβ-peptides and neurofibrillary tangles containing hyperphosphorylated tau protein. The formation ofneurofibrillary tangles is thought to be promoted by amyloid formation and is why the cellular events surrounding the formation and interactionsof the Aβ-peptide is a prime target for Alzheimer’s research. In this thesis, the gene of the highly aggregation prone form of Aβ-peptide, the Aβ1-42, has been inserted in a Drosophila melanogaster to promote expression in the central nervous system through the use of the Gal4-UAS system.Gene expression analysis was done using a RNA purification kit, translating the RNA into cDNA using RT-PCR and the levels were analyzed usingquantitative real-time PCR. For protein expression analysis the immunological techniques of dot blot and western blot were used combined withan immunoprecipitation step using magnetic beads. A fibrillation experiment was also performed to look into the potential seeding effect onamyloid formation from the Aβ1-42 expressing Drosophila using fluorescence spectroscopy.The aim for this thesis was to look into expression of the Aβ1-42 gene and the impact of ageing on expression levels. Another aim was to try andseparate and detect soluble Aβ-peptide species from tissue homogenates of Drosophila.No amplification could be detected in the quantitative real-time PCR, most likely due to concentration issues of the reaction components. For thisreason gene expression could never be quantified nor could the effect of ageing and gene expression be looked into. Insoluble aggregates but nosoluble Aβ-peptide species could be detected or separated from the tissue of the Drosophila. No seeding effect on the amyloid formation could bestatistically determined by the fibrillation experiment, but interesting quenching effects on the total quantum yield of Aβ fibrils in the presence ofbrain homogenates were noted.

In insects, hemocytes are considered as the only source of plasma prophenoloxidase (PPO). PPO also exists in the hemocytes of the hematopoietic organ that is connected to the wing disc of Bombyx mori. It is unknown whether there are other cells or tissues that can produce PPO and release it into the hemolymph besides circulating hemocytes. In this study, we use the silkworm as a model to explore this possibility. Through tissue staining and biochemical assays, we found that wing discs contain PPO that can be released into the culture medium in vitro. An in situ assay showed that some cells in the cavity of wing discs have PPO1 and PPO2 mRNA. We conclude that the hematopoietic organ may wrongly release hemocytes into wing discs since they are connected through many tubes as repost in previous paper. In wing discs, the infiltrating hemocytes produce and release PPO probably through cell lysis and the PPO is later transported into hemolymph. Therefore, this might be another source of plasma PPO in the silkworm: some infiltrated hemocytes sourced from the hematopoietic organ release PPO via wing discs.

In nature, all organisms face the daily challenges created by a fluctuating environment. Circadian clocks synchronize behaviour and physiology allowing an organism to adapt to and predict daily changes to environmental conditions. In the fruit fly, Drosophila melanogaster, circadian clocks reside in a set of ~150 neurons in the brain, collectively referred to as the central clock, and in the cells of many peripheral tissues. The central clock regulates daily behavioural rhythms, whereas peripheral clocks are thought to regulate the local metabolic activities of the cells in which they reside. In this thesis, I demonstrate that a peripheral clock resides in the abdominal fat body, a tissue analogous to the mammalian liver and adipocytes. Moreover, I show that flies display a temporal feeding pattern that is partly regulated by a peripheral clock. I propose that the central clock and peripheral clocks coordinate to regulate the timing of fly feeding behaviour.

Foraging behaviour in Drosophila melanogaster larvae is influenced by natural allelic variation in the foraging (for) gene that encodes a cyclic GMP – dependent protein Kinase (PKG), such that rovers (forR) traverse greater distances while foraging than sitters (fors). Foraging behaviour is also influenced by natural allelic variation in the wings up A (wupA) gene that encodes the Troponin-I protein (TnI). Specifically, wupAlow allele suppresses the dominance of the forR allele, turning rovers into sitters. The dominance of the natural wupA alleles and their interactions with allelic combinations in for has not been characterized. I conducted various crosses and found that wupA alleles exhibit incomplete dominance. More importantly, I found that allelic combinations of wupA and for gave rise to a range in larval foraging behaviour. In this study, I propose that this gradient effect in foraging behaviour is due to variation in levels of PKG activity and TnI phosphorylation potential.

In nature, all organisms face the daily challenges created by a fluctuating environment. Circadian clocks synchronize behaviour and physiology allowing an organism to adapt to and predict daily changes to environmental conditions. In the fruit fly, Drosophila melanogaster, circadian clocks reside in a set of ~150 neurons in the brain, collectively referred to as the central clock, and in the cells of many peripheral tissues. The central clock regulates daily behavioural rhythms, whereas peripheral clocks are thought to regulate the local metabolic activities of the cells in which they reside. In this thesis, I demonstrate that a peripheral clock resides in the abdominal fat body, a tissue analogous to the mammalian liver and adipocytes. Moreover, I show that flies display a temporal feeding pattern that is partly regulated by a peripheral clock. I propose that the central clock and peripheral clocks coordinate to regulate the timing of fly feeding behaviour.

Foraging behaviour in Drosophila melanogaster larvae is influenced by natural allelic variation in the foraging (for) gene that encodes a cyclic GMP – dependent protein Kinase (PKG), such that rovers (forR) traverse greater distances while foraging than sitters (fors). Foraging behaviour is also influenced by natural allelic variation in the wings up A (wupA) gene that encodes the Troponin-I protein (TnI). Specifically, wupAlow allele suppresses the dominance of the forR allele, turning rovers into sitters. The dominance of the natural wupA alleles and their interactions with allelic combinations in for has not been characterized. I conducted various crosses and found that wupA alleles exhibit incomplete dominance. More importantly, I found that allelic combinations of wupA and for gave rise to a range in larval foraging behaviour. In this study, I propose that this gradient effect in foraging behaviour is due to variation in levels of PKG activity and TnI phosphorylation potential.

Populations where three different lethals are segregating as alleles have been analysed for the conditions of equilibrium and for the trends during elimination of one allele. Early and late selection as well as sexdependent and sexindependent selection has been taken into consideration.Cage populations of Drosophila melanogaster with different fourth chromosome lethals have been followed and compared to the theoretical model. When two marker chromosomes (ciDpol and spaCat) and one out of four recessive lethal chromosomes l(4)5, 1(4)8, 1(4)10 or l(4)14 were used, the same marker chromosome (ciDpol) became extinct in all populations. Early and late selective values which were obtained directly from the populations were compared to estimates of fitness components obtained in specially designed experiments of viability, developmental rate, mating ability and fecundity. When two out of the four recessive lethals and the marker chromosome, spaCat , were combined in new populations, all populations attained equilibrium withôut extinction. A correlation was found between the time of death of the lethal homozygotes and the equilibrium genotype frequencies. Overall selective values at equilibrium were estimated. / digitalisering@umu

Apicobasal polarity is essential for epithelia formation and maintenance. Cell junctions, namely the zonula adherens in Drosophila melanogaster, are the morphological landmarks that define and distinguish the apical from the basal surface. This resulting compartmentalisation is key for the cell and consequently the epithelia. To maintain proper junctions, cells make use of several protein complexes and their interactions. Among these complexes, the Crumbs (Crb) network stands out. Mutations in Crumbs (crb11A22) lead to zonula adherens collapse, consequent loss of apical surface and disaggregation of the epithelia. However, the mechanisms behind this are not known and havenʼt been addressed using modern techniques such as live imaging. Several things came out of the dataset obtained from the FRAP experiments. Firstly, protein kinetics are better described when a double exponential fit curve is used, which raises the possibility that two cell processes might be involved in the recovery observed for the different markers. Another finding was the fact that the kinetics of some polarised protein markers is not the same in every region of the embryo. Distinct areas of the embryo with different morphogenetic activity levels show different kinetics for the same compartment marker. That was the case with SpiderGFP (whole plasma membrane marker) and SASVenus (apical plasma membrane marker) where τ2 was lower in the posterior region of the embryo which is characterised by intense cell movements resulting from convergence extension. DE-CadGFP (zonula adherens marker) and lacGFP (basolateral marker) behaved similarly in the whole embryo. This indicates that convergence extension shows different trafficking needs for the apical surface. In crb11A22, SpiderGFP kinetic spatial differences were not observed. τ2 in the anterior (low level of morphogenesis) is affected and similar to wild type τ2 levels in the posterior. This could pinpoint the fact that the epithelia disaggregation is a result of trafficking failure of apical components. Live imaging of DE-CadGFP in crb11A22 background revealed initial disaggregation in the anterior part of the embryo, which strengthens the idea that Crb is required for adherens junction stabilisation and maintenance.