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Role of adipokinetic hormone during starvation in Drosophila
KUTHANOVÁ, Michaela
The role of adipokinetic hormone (Drome-AKH) in maintaining the levels of basic nutrients, under starvation conditions, was studied using Drosophila melanogaster mutants with AKH deficiency (Akh1) and AKH abundance (EE-Akh). Our results showed lipids as the main energy reserve in Drosophila, and their physiological level and metabolism were shown to be under the control of AKH. AKH abundance in the body resulted in lower levels of triacylglycerols and diacylglycerols than in the controls, probably due to a more intensive metabolism; interestingly, there was a disproportional representation of fatty acids in triacylglycerols and diacylglycerols in Drosophila. Lower level of glycogen and its partial control by AKH suggest its lesser role as the storage substance. However, maintenance of free carbohydrate level in Drosophila seemed to be critical; when glycogen stores are exhausted, carbohydrates are synthesized from other sources. Protein levels and their alterations, under starvation, did not seem controlled by AKH. AKH-deficient flies were more resistant while AKH-abundant flies were more sensitive to starvation; females were found to be more resistant than males, regardless of the AKH level, probably due to higher body mass and higher amount of nutrients. However, in accordance with the level of all nutrients, that of AKH also gradually decreased with prolonged starvation
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Vliv adipokinetického hormonu na působení jedu z vosičky, \kur{Habrobracon hebetor}, ve svalové tkáni octomilky, \kur{Drosophila melanogaster}
PODLAHOVÁ, Šárka
Adipokinetický hormon (AKH) zpožďuje nebo zmírňuje nervosvalovou paralýzu způsobenou jedem z vosičky, Habrobracon hebetor, avšak mechanismus působení zatím není zcela objasněn. Tato práce detailně popisuje metodiku přípravy polotenkých a ultratenkých řezů, analyzovaných pomocí transmisní elektronové mikroskopie, která umožňuje detailní pozorování změn ve svalové ultrastruktuře thoraxu Drosophila melanogaster po ošetření vosičkovým jedem a AKH. Výsledky odhalily, že vosičkový jed způsobuje výrazné změny v ultrastruktuře myofibril a mitochondrií, zatímco po aplikaci jedu společně s AKH byly změny potlačeny. Samotný AKH neovlivnil strukturu svalových vláken, avšak vyvolal vakuolizaci mitochondrií. Mimoto ošetření vosičkovým jedem vedlo ke značnému prodloužení sarkomer.
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Study of adipokinetic hormone role in insects stressed by entomopathogenic nematodes
IBRAHIM, Emad Ahmed Sayed
In this thesis, the effect of infection elicited by entomopathogenic nematodes (EPNs) Steinernema carpocapsae on Pyrrhocoris apterus and Drosophila melanogaster models were evaluated, and a role of adipokinetic hormones (AKHs) during the infection was characterized. These were monitored by determination of mortality, and various biochemical and physiological characteristics such as AKH levels both in the central nervous system (CNS) and in hemolymph, AKH gene expression in CNS, level of anti-oxidative stress markers, general metabolism and level of nutrients in normal and genetically modified insects. At P. apterus the mortality tests revealed that application of AKH increases the efficacy of EPN treatment. This result was confirmed using the firebugs with AKH receptor deficiency. Further, the increase of AKH expression and AKH levels in CNS and hemolymph seemed to be coordinated after the nematode treatment. At the D. melanogaster model also, the effect of adenosine into the above-mentioned characteristics was included. For this, mutants in AKH (AHK1), adenosine receptor (AdoR1) genes, and in both these genes together (AHK1 AdoR1 double mutant) were employed. Altogether, the results confirmed the involvement of AKH, and partially also adenosine into the antistress defense reactions elicited by the nematobacterial infection. Finally, the last part of the study was focused on examination of the vitellogenin (Vg) role in the defense reaction in firebug body P. apterus affected by two entomopathogenic organisms, the nematode S. carpocapsae and the fungus Isaria fumosorosea. The results revealed that Vg proteins play an important role in the defense against both types of the infections and are also able to kill entomopathogenic bacteria Xenorhabdus nematophila, that are symbionts of S. carpocapsae and that increase toxicity of this nematode.
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Úloha adipokinetického hormonu v metabolismu základních živin u octomilky obecné \kur{Drosophila melanogaster}
KUTHANOVÁ, Michaela
Tématem této práce bylo posoudit různé metabolické charakteristiky u octomilky obecné Drosophila melanogaster s produkcí deficientního AKH a s poruchou v adenosinovém receptoru.Hlavním úkolem bylo prostřednictvím experimentů posoudit zapojení AKH a adenosinu dokontroly metatabolických drah. Byla stanovena hladina základních živin, hladina Drome-AKH, mortality a další charakteristiky u octomilky obecné během hladovění. Bylo zjištěno, že AKH u octomilky obecné ovlivňovala metabolizmus zásobních živin, nicméně úloha adenosinu zůstala nejasná.
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Anti-oxidative stress response in Drosophila melanogaster - The role of adipokinetic hormone and adenosine
ZEMANOVÁ, Milada
In this thesis, the phenomena of the oxidative stress (OS) and anti oxidative stress responses in insects are described in a comprehensive review, and the outcomes of the experimental work are presented. The focus of the work was on defence reactions and their putative control by the adipokinetic hormone (AKH) and adenosine in the Drosophila melanogaster model. For this purpose, we studied the effect of the paraquat (oxidative stressor) treatment on adult flies and larvae carrying mutations in Akh (Akh1) and adenosine receptor (AdoR1) genes, and in both these genes together (Akh1 AdoR1 double mutant). The initial mortality tests revealed the double mutant Akh1 AdoR1 was more sensitive to OS than either of the single mutants. The AKH synthesis under the OS condition seems to be out of the gene expression control, since the increase of an AKH amount in CNS was not linked with the stimulation of Akh gene expression after a paraquat treatment. Further, the gene expression of the antioxidant enzyme glutathione S transferase D1 (GstD1) increased rapidly with OS, though the enzyme activity increased negligibly regardless of both the OS and mutations. Interestingly, the relative expression of GstD1 gene was minimal in the double Akh1 AdoR1 mutant; thus, it was concluded that both AKH and adenosine are employed in the GstD1 gene expression control. Similarly, AKH and adenosine seem to act in tandem in glutathione (GSH) regeneration, since the GSH level was significantly lower in all untreated deficient flies with the maximal effect in the Akh1 AdoR1 double mutant; accordingly, the reduction in the GSH level was enhanced by paraquat treatment. Altogether, the important roles of both AKH and adenosine in the anti oxidative stress response in D. melanogaster were demonstrated.
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Mode of action of adipokinetic hormone at the sub-cellular level in potentiating anti-oxidative responses in insects.
BEDNÁŘOVÁ, Andrea
Adipokinetic hormones (AKHs) are neuropeptides from the arthropod AKH/RPCH (adipokinetic hormone/ red pigment concentrating hormone) family. The typical AKH is an octa-, nona- or decamer that is synthesized, stored and released by the neurosecretory cells of the corpora cardiaca (CC) connected to the brain and primarily involved in the mobilization of energy reserves from the fat body in insects. In addition to its well established role in energy metabolism, AKH has also been implicated to be involved in stress responses specifically to oxidative stress. Oxidative stress induced elevation of AKH levels as well as a modulation of biomarkers of oxidative damage following exogenous application of AKH have been demonstrated. However, the discrete steps involved in the mode of action of AKH in triggering an anti-oxidative response is far from clear. Given the role of AKH as a neuroendocrine factor that mediates a response to oxidative stress, the mode of action of AKH at the sub-cellular level was investigated. Using isolated central nervous system (brain) as an in vitro model, we establish that AKH can potentiate an anti-oxidative response to oxidative stress. Further, we also demonstrate that AKH uses a conserved signal transduction mechanism involving both protein kinase C (PKC) and cyclic adenosine monophosphate (cAMP) and by mobilizing both intra as well as extra-cellular Ca2+ stores to elaborate its anti-oxidative response. Finally, using the genetically tractable fruit fly Drosophila melanogaster, we demonstrate through RNAi mediated knockdown of AKH synthesis as well as overexpression of AKH using the GAL4/UAS system, that the fork-head box transcription factor (dFoXO) might function downstream of AKH signaling in its stress responsive role. These results implicate AKH as a stress hormone while offering possibilities to further identify specific regulatory mechanisms and downstream effector molecules. Since stress signaling pathways are conserved, insights obtained from such studies on insects will offer some unique avenues for understanding stress responses and related pathologies in vertebrates including humans.
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Adipokinetic hormone counteracts oxidative stress elicited in insects by hydrogen peroxide: in vivo and in vitro study
BEDNÁŘOVÁ, Andrea
The involvement of members of Adipokinetic hormone (AKH) family in regulation of response to oxidative stress (OS) was investigated in the present study. OS was elicited in the insect body by exogenous application of hydrogen peroxide. In vivo experiments reveal that injection of hydrogen peroxide into the haemocoel of the firebug, Pyrrhocoris apterus, increases the level of AKH in the central nervous system (CNS) and in the haemolymph. The injection of hydrogen peroxide also increases the mortality of experimental bugs, whereas co-injection of hydrogen peroxide with Pyrap-AKH reduces mortality to control levels. Importantly, an increase in haemolymph protein carbonyl (an OS biomarker) levels is decreased to control levels when hydrogen peroxide is co-injected with Pyrap-AKH. Similar results were obtained using in vitro experiments. OS biomarkers such as malondialdehyde and protein carbonyls were significantly enhanced upon exposure of isolated CNS to hydrogen peroxide in vitro, whereas co-treatment of the CNS with hydrogen peroxide and Pyrap-AKH reduces their level significantly. Catalase activity was measured as well and the same pattern was observed. The results of the present study provide strong support on the feedback regulation between oxidative stressors and AKH action, and implicate AKH in counteracting OS.
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Role of adipokinetic peptides in control of insect anti-stress reactions
Kodrík, Dalibor
Adipokinetic hormones (AKHs) control predominantly insect metabolism, but are involved also in a number of other activities. Recently, it was proven that AKHs participated also in the activation of antioxidant protection mechanisms: they increased a level of reduced glutathione and additionally, modulated the activity of catalase that reduces hydrogen peroxide to water and oxygen. It has also been found that AKHs participated on antistress reaction elicited by insecticides and increased the lethal effect of the insecticides alone. The results show that AKHs provide an initiation of the complex antistress response that involves both biochemical and physiological protective mechanisms.
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Contribution to the study of insect stress hormones
ALQUICER BARRERA, Glenda Paola
The present study considers the versatile role of adipokinetic hormones (AKHs). Using the firebug Pyrrhocoris apterus as a model insect, the hypothesis that AKHs mediate stress response mechanisms was explored. The outcome indicated that there is a positive feedback regulation between an oxidative stressor action and the level of AKH in insect body, and that AKHs might be involved in the activation of antioxidant protection mechanism. Further results revealed a functional homology between AKH and the mammalian hormone glucagon. The possible effects of glucagon on mobilisation of energy reserves and on elicitation of an antioxidant response to oxidative stress were investigated. As a result, glucagon-immunoreactive material was detected for the first time in the firebug central nervous system and gut. Antioxidant mechanisms are elicited after glucagon treatment but it did not involve mobilization of energy reserves or AKH level changes. As a complement, the existence of a feedback between juvenile hormone and AKH was investigated by topical application of the juvenile hormone analogue methoprene, which influenced the release of AKH from the central nervous system into the haemolymph and induced a partial reduction of lipid content in haemolymph.
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