| 1. |
- Dziedziech, Alexis, 1991-
(författare)
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Timing Matters : Wounding and entomopathogenic nematode infection kinetics
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Over time, insects have developed complex strategies to defend themselves against presenting threats. However, in the evolutionary arms race of survival, pathogens have adapted to quickly overcome the immune response mounted by the host. In this thesis, we assess how quickly entomopathogenic nematodes (EPNs) can overcome the host, Drosophila melanogaster. We then look at the clotting reaction at a hypothetical point of entry for the nematode and bring resolution to the order of protein interaction focusing on three proteins important in the anti-nematode defense. Finally, we look closer into detail at how crystal cells secrete one of those proteins, prophenoloxidase (PPOII) using a mode of programmed cell death. (Paper I) In the course of EPN infection, little was known about how quickly the worms can overcome the host immune system. Here we found that after penetrating the host, EPNs cause septicemia within 4 to 6 hours. (Paper II) Three proteins, Glutactin (Glt), Transglutaminase (Tg), and PPOII have been found to be important in the anti-nematode response. Here we created GFP-tagged fly constructs to follow their role in clot formation. In early clot formation, Tg was immediately secreted from hemocytes though it was localized around the cell membrane, Glt then entered clot fibers followed by PPOII which acted in late clot formation. (Paper III) Here we looked closer into Tg and PPOII secretion variability. PPOII from immature, but not mature crystal cells colocalized with a membrane marker. Tg, when driven with a pan tissue driver, was found located in clotting fibers, in contrast with paper II. (Paper IV) In an in vivo immune scenario, crystal cells were recruited to the wound site and burst rapidly in a caspase-dependent manner. We demonstrate that the mode of programmed cell death, pyroptosis, exists in Drosophila by way of convergent evolution.This thesis brings to light the variation found within the infection process for EPNs as well as the clotting response based on larval age, tissue type, and the maturity of a single cell type. Timing in each of these immune scenarios can give very different indications about the kind of immune response mounted and even the role of an individual cell.
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| 2. |
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| 3. |
- Khalili, Dilan, 1988-
(författare)
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Stress and immune signaling in a Drosophila tumor model
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cancer cells contain multiple biological alterations that allow them to escape from host surveillance mechanisms. One of the mechanisms that play an essential role in host protection against tumor growth is immunity. However, the immune system may act as a double-edged sword with the potential to both promote and limit tumor growth in a context-dependent manner. This involves both internal and external signaling events such as stress signaling pathways but also communication between cells and/or between cells and the extracellular matrix (ECM). In this thesis, Drosophila melanogaster (the fruit fly) was used to understand the role of two immune-related components, namely the antimicrobial peptide Drosomycin (Drs) and a chitinase-like protein (Idgf3), in a tumor model that involves a tubular organ, namely the salivary glands.In Paper I we investigated Drs function and regulation upon expression of the oncogene RasV12. Initially, Drs was upregulated in the whole SG upon RasV12 expression. However, at the later stage of the tumor, Drs expression was restricted to the proximal region. In contrast, at the distal region, the hallmarks of cancer phenotypes, such as activation of the pro-tumorogenic JNK pathway, adhering immune cells and production of reactive oxygen species (ROS), were elevated. By overexpressing Drs in the distal region, we found that Drs interferes with most cancer hallmarks, including the JNK-pathway, recruitment of immune cells, and ROS production.In Paper II we further characterized the hallmarks of cancer in our model system by addressing external and internal changes and whether Drs may influence them. At the extracellular compartment, we demonstrate the redistribution of the ECM in tumors, recruitment of immune components, including prophenoloxidases (PPOs) and Drs, and identified F-actin as a part of the ECM. Intracellularly, the organs' primary function, secretion, is lost, and the cell’s epithelial organization is disturbed. Drs reversed the majority of these changes.In Paper III we addressed the role of Idgf3 and its effect on external and internal cues. Initially, we found that Idgf3 was induced in the RasV12 salivary glands. Upon knock-down of Idgf3, the cellular organization was restored, and tumor growth was limited. Moreover, Idgf3 expression was correlatively increasing with the progression of the tumor. In line with Paper I, we found a similar correlation with the JNK pathway. Through genetic experiments, we show JNK-mediated regulation of Idgf3 through ROS. By addressing the subcellular localization of Idgf3, we found the protein internalized within enlarged vesicles, which were coated with a cytoskeletal protein, Spectrin. Furthermore, the formation of enlarged vesicles promoted tumor progression through loss of cellular organization. Taken together, the findings presented here emphasize the complexity of the immune system and its function in tumor progression. Further studies are necessary to understand the potential for tumor therapy.
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| 4. |
- Arefin, Md. Badrul, 1983-
(författare)
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Molecular characterization of the Drosophila responses towards nematodes
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- A sophisticated evolutionary conserved innate immune system has evolved in insects to fight pathogens and to restrict damage in harmful (danger) situations including cancer. A significant amount of knowledge about different infection models in Drosophila has been generated in past decades, which revealed functional resemblances and implications for vertebrate systems. However, how Drosophila responds towards multicellular parasitic nematodes and in danger situations is still little understood. Therefore, the aim of the thesis was to characterize multiple aspects of the host defense in the two important contexts mentioned above.We analyzed the transcriptome profiles of nematode-infected Drosophila larvae with uninfected samples. For this we employed the entomopathogenic nematode Heterorhabditis bacteriophora with its symbiont Photorhabdus luminescence to infect Drosophila larvae. We found 642 genes were differentially regulated upon infection. Among them a significant portion belonged to immune categories. Further functional analysis identified a thioester containing protein TEP3, a recognition protein GNBP-like 3, the basement membrane component protein Glutactin and several other small peptides. Upon loss or reduced expression of these genes hosts showed mortality during nematode infections. This study uncovers a novel function for several of the genes in immunity.Furthermore, we investigated the cellular response towards nematodes. When we eliminated hemocytes genetically (referred to as hml-apo) in Drosophila, we found hml-apo larvae are resistant to nematodes. Subsequent characterization of hml-apo larvae showed massive lamellocyte differentiation (another blood cell type which is rare in naïve larvae), emergence of melanotic masses, up- and down-regulation of Toll and Imd signaling respectively suggesting a pro-inflammatory response. Moreover, a striking defective leg phenotype in adult escapers from pupal lethality was observed. We identified nitric oxide (NO) as a key regulator of these processes. We also showed that imaginal disc growth factors 3 (IDGF3): (a) protects hosts against nematodes, (b) is a clotting component and (c) negatively regulates Wnt and JAK/STAT signaling. To follow larval behavior in the presence or absence of nematodes we monitored Drosophila larval locomotion behaviors using FIMtrack (a recently devised automated method) to elucidate evasive strategies of hosts. Finally, we characterized host defenses in three Drosophila leukemia models with and without nematode infection. Taken together, these studies shed light on host responses in two crucial circumstances, nematode infections and danger situations.
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| 5. |
- Bidla, Gawa, 1975-
(författare)
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Genetic and molecular dissection of hemolymph coagulation and melanization in Drosophila melanogaster
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Injury to epithelial barriers puts metazoans at risk of loss of body fluid and contamination of their body by foreign particles. This risk is even exacerbated in insects, which have an open circulatory system and as a result, quickly need to seal wounds in order to keep a fairly constant internal milieu. Due to paucity of information on biochemical and molecular basis of insects’ clot, we studied how hemolymph of Drosophila melanogaster forms a clot, leading to a better understanding of responses after injury or infection in flies. By comparing hemolymph of Drosophila after bleeding with that described for an earlier model Galleria mellonella, we showed that a bona fide clot forms in Drosophila. The Drosophila clot is a fibrous network of crosslinked hemolymph proteins, which incorporates blood cells (plasmatocytes) extending shorter cellular processes of filopodia compared to cells outside the clot. Also, some plasmatocytes in the clot show features of apoptotic death while other blood cells (crystal cells) quickly rupture. The clot sequesters bacteria, as bacteria tethered to clot did not move. Clotting factors isolated include, Hemolectin (Hml) previously implicated in clotting, the immune induced protein Fondue and hemolymph proteins such as apolipophorin 2, fat body protein 1 and larval serum protein 1 γ. Hml mutants were more susceptible to infections when tested in a genetically sensitized background, suggesting that the clot may contribute to innate immunity. Clot also formed in hemolymph without phenoloxidase, an enzyme required for melanization and previously thought to be important for clot formation. However, we found that PO activity strengthens the clot to form a more solid plug. We found PO activity in clot to be induced in a transcription independent manner by inner membrane phospholipids: phosphatidylserine (PS) and phosphatidylinositol (PI) exposed on dead plasmatocytes and ruptured crystal cells. This is in contrast to induction of the enzyme during infection, which requires microbial components and transcriptional induction. However, both activation of PO in the clot and activation after infection appear to depend on proteases. Surprisingly, neither PS nor PI induced PO activity in the lepidopteran Galleria mellonella, in which the enzyme activity was instead induced by the microbial components peptidoglycan. This result may caution against generalizations of findings from using only one particular insect species. Finally, we found that the rupture of crystal cell during clot formation requires the Drosophila TNF homologue Eiger, JNK homologue Basket and small GTPases. This work therefore adds hemolymph clotting to the responses after injury or infection in flies and largely establishes Drosophila as a model to study coagulation of insect hemolymph. This will lead to a more comprehensive picture of Drosophila immunity with implications for other innate immune systems including our own.
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| 6. |
- Krautz, Robert, 1986-
(författare)
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Drosophila immune responses in a model for epithelial hypertrophy
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Apoptosis, differentiation and proliferation have to be tightly balanced and thus regulated to maintain tissue homeostasis. Stress, metabolic cues, genetic variability, infections and physiological host-commensal interactions influence this balance and thus need to be integrated. Therefore, beyond the discrimination between self and non-self (i.e., foreign) also damage inflicted on tissues under sterile conditions is perceived by the immune system due to altered tissue integrity. Growing knowledge of the interaction between the immune system and wounded or more generally altered tissues allows inferring on anti-tumorous immune responses, too. Despite the lack of adaptive immunity, Drosophila mounts solid and versatile innate immune responses that functionally and molecularly share many properties with their vertebrate counterparts. In fact, tissue overgrowth, tissue dysplasia or endogenous danger signaling activate systemic Toll-signaling in the fat body indicating a role for the Drosophila immune system in maintaining tissue homeostasis.Here we characterize systemic and local immune responses towards altered or transformed tissues by using a Drosophila hypertrophy model, which is based on the overexpression of a dominant-active variant of the small GTPase Ras (Ras85DG12V) in salivary glands and wing discs. We characterized the strong induction of hemocyte recruitment to the glands as a consequence of JNK-dependent MMP1-expression and basal membrane degradation. Apart from this cellular immune reaction, transcriptome profiling revealed comprehensive humoral immune responses mounted by the fat body that involved signatures of Toll- and imd-activation. Moreover, a novel tissue-autonomous response that was spatially restricted to the anterior end of the RasV12-expressing salivary gland itself was identified. While multiple immune genes were found to be upregulated in the anterior compartment as detected by RNA sequencing, particular focus was given to the effector peptide Drosomycin (Drs). Overexpression of Drs with RasV12 in the entire gland similar to the inhibition of the JNK-pathway was able to selectively rescue a characteristic set of RasV12-induced phenotypes, which ultimately blocks the recruitment of hemocytes. Thereby, local immune-related responses in RasV12-expressing salivary glands are able to restrict the tissue damage induced by hypertrophic growth.
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| 7. |
- Lindgren, Malin, 1975-
(författare)
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Molecular and functional characterization of the insect hemolymph clot
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- All metazoans possess an epithelial barrier that protects them from their environment and prevents loss off body fluid. Insects, which have an open circulatory system, depend on fast mechanism to seal wounds to avoid excessive loss of body fluids. As in vertebrates, and non-insect arthropods such as horseshoe crab and crustaceans, insects form a clot as the first response to tissue damage. Insect hemolymph coagulation has not been characterized extensively at the molecular level before, and the aim of my studies was to gain more knowledge on this topic. Morphological characterization of the Drosophila hemolymph clot showed that it resembles the clots previously described in other larger bodied insects, such as Galleria mellonella. The Drosophila clot is a fibrous network of cross-linked proteins and incorporated blood cells. The proteins building up the clot are soluble in the hemolymph or released from hemocytes upon activation. Since bacteria are caught in the clot matrix and thereby prevented from spreading it is likely that the clot serves as a first line of defense against microbial intruders. The bacteria are not killed by the clot. What actually kills the bacteria is not known at this point, although the phenoloxidase cascade does not seem to be of major importance since bacteria died in the absence of phenoloxidase. We identified and characterized a new clot protein which we named gp150 (Eig71Ee). Eig71Ee is an ecdysone-regulated mucin-like protein that is expressed in salivary glands, the perithophic membrane of the gut and in hemocytes, and can be labeled with the lectin peanut agglutinin (PNA). Eig71Ee was found to interact with another clot protein (Fondue), and the reaction was catalyzed by the enzyme transglutaminase. This is the first direct functional confirmation that transglutaminase acts in Drosophila coagulation. A protein fusion construct containing Fondue tagged with GFP was created. The fusion construct labeled the cuticle and the clot, and will be a valuable tool in future studies. Functional characterization of the previously identified clotting factor Hemolectin (Hml) revealed redundancy in the clotting mechanism. Loss of Hml had strong effects on larval hemolymph clotting ex vivo, but only minor effects, such as larges scabs, in vivo when larvae were wounded. An immunological role of Hml was demonstrated only after sensitizing the genetic background of Hml mutant flies confirming the difficulty of studying such processes in a living system. Hemolectin was previously considered to contain C-type lectin domains. We reassessed the domain structure and did not find any Ctype lectin domains; instead we found two discoidin domains which we propose are responsible for the protein’s lectin activity. We also showed that lepidopterans, such as Galleria mellonella and Ephestia kuehniella, use silk proteins to form clots. This finding suggests that the formation of a clot matrix evolved in insects by the co-option of proteins already participated in the formation of extracellular formations.
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| 8. |
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| 9. |
- Wang, Zhi, 1979-
(författare)
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Functional study of hemolymph coagulation in Drosophila larvae
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Many pathogen infections in nature are accompanied by injury and subsequent coagulation. Despite the contribution of hemolymph coagulation to wound sealing, little is known about its immune function. Based on the molecular knowledge of Drosophila innate immunity, this thesis investigated the immune function of clot both in vitro and in vivo, the immune relevant genes involved in a natural infection model, involving entomopathgenic nematodes (EPN) and the factors leading to crystal cell activation. Transglutaminase (TG) and its substrate Fondue (Fon) have been identified as bona fide clot components in Drosophila larvae. By knocking down TG or Fon via RNAi, we observed an increased susceptibility to EPN in larvae. In addition, this increased susceptibility was associated with an impaired ability of hemolymph clots to entrap bacteria. Immunostaining revealed that both clot components (Fon and TG) were able to target microbial surfaces. All these data suggest an immune function for the Drosophila hemolymph clot. Strikingly, similar results were obtained when we ran parallel experiments with human FXIIIa, an ortholog of Drosophila TG, indicating a functional conservation. We also found evidence for the regulation on both clot and immunity by eicosanoids in Drosophila larvae. The combination of EPN infection with the Drosophila model system allowed us to discover an immune function for TEP3 and Glutactin. However the molecular mechanism underlying the involvement of these two proteins in this particular host-pathogen interaction remains to be elucidated. Prophenoloxidase, the proform of enzyme involved in hardening the clot matrix, has been shown to be released by rupture of crystal cells. This cell rupture is dependent on activation of the JNK pathway, Rho GTPases and Eiger. Our work further identified the cytoskeletal component, Moesin, and the cytoskeletal regulator Rac2 as mediators of cell rupture. Despite the possible role of caspases in crystal cell activation, such cell rupture was turned out to be different from apoptosis. The implication of Rab5 in this process indicated that proper endocytosis is required for cell activation and subsequent melanization. Our findings furthered not only our understanding of the release of proPO via cell rupture but also our knowledge on different paths of immune cell activation.
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| 10. |
- Wilhelmsson, Christine, 1973-
(författare)
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Proteomics of the Drosophila hemolymph clot and the function of transglutaminase
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Insects rely on a fast and effective coagulation and wound response to avoid loss of body fluids and immobilize pathogens. Arthropod coagulation is in some respect equivalent to vertebrate coagulation but most factors and the regulation of coagulation systems seem not to be phylogenetically conserved. To get a more complete picture of insect clotting we studied the molecular and functional nature of Drosophila hemolymph coagulation. We developed new proteomic methods to collect Drosophila clotting factors. Several candidate factors were identified, including both predicted and novel clot proteins. Five putative TG (transglutaminase) substrates were found and we could also demonstrate that the clot is involved in immobilization of bacteria. Further investigating the role of TG we found TG to be important for Drosophila coagulation and that Fondue is a major substrate of the enzyme. Using fon RNAi knockdown we showed that Fondue affects the physical properties of the clot. A fon-GFP fusion construct was generated to follow its expression. The cuticle and the clot were labelled suggesting that Fondue is incorporated into both cuticle and clot. Clot properties and composition were affected by inhibiting TG chemically (MDC) and genetically (RNAi). Moreover, interaction between Fondue and Eig71Ee was demonstrated. Previous results indicated that coagulation could have an immune function. In hemolymph preparations, containing selected microorganisms, small deposits were seen on the microbial surfaces. The contents of these were investigated, revealing the presence of procoagulants. The targeting of microbes is instant and depends on TG and its substrates. Entomopathogenic nematode infections were performed to validate the functional importance of TG. TG RNAi knockdown larvae showed increased mortality, supporting an immune function for TG. Altogether, our data provide a more comprehensive picture of Drosophila immunity, and may further improve the understanding of innate immunity in general.
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