Viruses are very abundant in water and hence diseases caused by viruses are common in marine organisms. These diseases create great problems for the commercial farming of crustaceans and mussels. One of the most common and most disastrous diseases for shrimp is caused by the white spot syndrome virus (WSSV), which is spread all around the world and also is infecting many different species of crustaceans including freshwater crayfish. Although during recent years knowledge has been gathered on the ways in which invertebrates defend themselves against bacteria and fungi virtually nothing is known about the defence processes elicited by virus. The aim of this work was to develop a model to use for studies of virus-host interactions in vivo and in vitro. Temperature was found to be important for the virus infectivity and at lower temperature the virus apparently did not replicate, but if animals kept at low temperature for more than 40 days were transferred to higher temperatures they died quickly due to an increased virus replication. In crayfish infected with the virus it was found that hemocytes did not degranulate and the melanization reaction was also inhibited in the hemocyes. Thus it is apparent that this virus interacts with the immune system and hemocytes in particular and to be able to study this in some greater detail it was necessary to develop a cell culture to study virus-host interactions at the molecular level. Hence, we have developed a stem cell culture from the hematopoietic tissue (hpt) that will differentiate and mature into hemocytes and which can be used to replicate the WSSV in the presence of an endogenous cytokine, astakine. Astakine is the first cytokine like-factor described which is directly involved in hematopoiesis in an invertebrate.

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.

Η παρούσα μελέτη διερευνά τη συμμετοχή της κινάσης της τριφωσφορικής ινοσιτόλης (PI3-kinase) στο σηματοδοτικό μονοπάτι που επάγεται παρουσία μικρομοριακών συγκεντρώσεων καδμίου (Cd 50 μΜ) σε αιμοκύτταρα του μυδιού Μ. galloprovincialis. Η μελέτη πραγματοποιήθηκε με χρήση της ουσίας wortmannin (wort), που αποτελεί αναστολέα της δραστικότητας της PI3-kinase. Σύμφωνα με τα αποτελέσματα, αιμοκύτταρα που επωάστηκαν για 1 ώρα σε διαφορετικές συγκεντρώσεις wortmannin παρουσίασαν σημαντική μείωση της βιωσιμότητάς τους σε συγκεντρώσεις μεγαλύτερες από 50 nM. Επιπρόσθετα, κύτταρα που προ-επωάστηκαν με μη- τοξικές συγκεντρώσεις wortmannin, πριν την έκθεσή τους σε Cd 50 μΜ, παρουσίασαν σημαντική μείωση της ικανότητας του μετάλλου να προκαλεί αύξηση της θνησιμότητας των κυττάρων, παραγωγή σουπεροξειδικών ανιόντων (.O2 -) και νιτρικών οξειδίων (ΝΟ), καθώς και να επάγει την λιπιδική υπεροξείδωση. Παράλληλα, κύτταρα που επωάστηκαν με τον φωρβολεστέρα PMA (10 μg/ml), που αποτελεί σημαντικό αγωνιστή της πρωτεϊνικής κινάσης C (PKC) η οποία ευθύνεται για την επαγωγή της διαδικασίας της αναπνευστικής έκρηξης (παραγωγή .O2 - και ΝΟ, μέσω της ενεργοποίησης της NADPH οξειδάσης και ΝΟ συνθετάσης αντίστοιχα) έδειξαν σημαντική αύξηση των επιπέδων (.O2 -) και ΝΟ, συγκριτικά με τα κύτταρα ελέγχου. Η παρουσία της wortmannin (1 και 50 nM) σε κύτταρα που εκτέθηκαν στον φωρβολεστέρα PMA ανέστειλε σημαντικά της ικανότητα του τελευταίου να προκαλεί αύξηση των επιπέδων ΝΟ, ενώ τα επίπεδα (.O2 -) παρέμειναν υψηλά. Από τα αποτελέσματά μας μπορούμε να συμπεράνουμε ότι η PI3- kinase μπορεί να σχετίζεται με μια PKC-ανεξάρτητη επαγωγή της δραστικότητας της NO συνθετάσης, ενώ η αλληλεπίδρασή της με την PKC φαίνεται να οδηγεί στην επαγωγή της δραστικότητας της NAPDH οξειδάσης. / The involvement of PI3-kinase in cadmium (Cd) mediated oxidative effects on hemocytes of mussel M. galloprovincialis was investigated. According to the results, hemocytes pre-treated for 15 min with non-toxic concentrations of wortmannin (1 and 50 nM, as revealed by neutral red retention assay), a specific PI3-kinase inhibitor, showed a significant attenuation of Cd ability (at concentration of 50 μΜ) to promote cell death, superoxide anions (.O2 -) production, nitric oxide (NO) generation and lipid peroxidation (MDA content) in any case. Furthermore, wort-treated cells showed a significant attenuation of PMA (10 μg/ml) ability to induce NO generation but not .O2 - production. It seems that PI3-kinase in cells faced with pro-oxidants, such as Cd, could lead to a PKC-independent induction of NO synthase activity, while the existence of a cross-linking among PI3-kinase and protein kinase C is seemed fundamental for the regulation of NAPDH oxidase activity, probably through a PKC-dependent signaling pathway.

Οι πολυφαινόλες είναι μια κατηγορία οργανικών χημικών ουσιών, που χαρακτηρίζονται από την παρουσία ενός ή περισσότερων υδροξυλίων, συνδεδεμένων με έναν ή περισσότερους αρωματικούς ή ετεροκυκλικούς δακτυλίους φαινόλης. Το ταννικό οξύ (ΤΑ), μια ειδική εμπορική μορφή της τανίνης, χρησιμοποιείται ευρέως στη βιομηχανία και αποτελεί μία από τις κυριότερες ουσίες των βιομηχανικών λυμάτων που διοχετεύονται στα υδάτινα οικοσυστήματα και στο έδαφος. Παρόλο το γεγονός ότι οι πολυφαινόλες δρουν αντιοξειδωτικά στα κύτταρα, η ισορροπία μεταξύ της αντιοξειδωτικής και της οξειδωτικής δράσης τους είναι υπό διερεύνηση. Η παρούσα μελέτη διερευνά τις αντιοξειδωτικές και οξειδωτικές επιπτώσεις του ταννικού οξέος σε αιμοκύτταρα του μυδιού Mytilus galloprovincialis, παρουσία τοξικών συγκεντρώσεων καδμίου. Συγκεκριμένα, αιμοκύτταρα που εκτέθηκαν σε διαφορετικές συγκεντρώσεις ΤΑ (1, 10, 20, 40 και 60 μΜ) για 1 h, έδειξαν σημαντική μείωση της βιωσιμότητάς τους, μόνο σε συγκεντρώσεις ΤΑ μεγαλύτερες από 40 μΜ. Παράλληλα, αιμοκύτταρα που εκτέθηκαν σε μικρομοριακές συγκεντρώσεις του μετάλλου (50 και 100 μΜ) έδειξαν σημαντική μείωση της βιωσιμότητάς τους. Προκειμένου να προσδιορίσουμε την αντιοξειδωτική ή οξειδωτική ικανότητα του ΤΑ, αιμοκύτταρα που είχαν προηγουμένως προ-επωαστεί σε διαφορετικές συγκεντρώσεις ΤΑ για 15 min, εκτέθηκαν σε μικρομοριακές συγκεντρώσεις του μετάλλου. Σύμφωνα με τα αποτελέσματά μας, κύτταρα που είχαν προ-επωαστεί σε συγκεντρώσεις ΤΑ 1- 40 μΜ, έδειξαν σημαντική αναστολή των οξειδωτικών επιπτώσεων του μετάλλου (παραγωγή σουπεροξειδικών ανιόντων/∙O2 −, οξειδίων του αζώτου/ ΝΟ, και προϊόντων λιπιδικής υπεροξείδωσης/ επίπεδα μηλονικής διαλδεϋδης), όσο και της ικανότητάς του να μειώνει τη βιωσιμότητα των κυττάρων, συγκριτικά με τις τιμές που μετρήθηκαν σε κύτταρα που εκτέθηκαν μόνο στο μέταλλο. Αντίθετα, σε κύτταρα που προ-επωάστηκαν σε ΤΑ 60 μΜ, πριν την έκθεσή τους στο μέταλλο, το ΤΑ εμφανίστηκε να δρα συνεργατικά με το μέταλλο. Τα αποτελέσματα της παρούσας μελέτης οδηγούν στο συμπέρασμα ότι το ΤΑ σε μικρομοριακές συγκεντρώσεις 1-40 μΜ μπορεί να δράσει ως ένας ισχυρός αντιοξειδωτικός παράγοντας, ενώ σε υψηλότερες συγκεντρώσεις μπορεί να προκαλέσει οξειδωτικές επιπτώσεις, ανάλογες με αυτές που προκύπτουν από ισχυρούς οξειδωτικούς παράγοντες, όπως τα κάδμιο. / Polyphenols are well-known organic substances, mainly characterized by the presence of one or more hydroxyl groups on one or more aromatic or heterocyclic phenol rings. Tannic acid (TA), a specific commercial form of tannin is a natural polyphenol, widely used in food, pharmaceutical, leather and chemical industry. It is one of the main organic compounds of industrial effluents discharged into aquatic ecosystems and soil, causing environmental pollution. Despite the fact that a lot of studies reported that polyphenols could act as antioxidants in different cells, the balance between their antioxidant and pro-oxidant properties remains still unclear. According to the later, the present study investigates the antioxidant and pro-oxidant potency of TA in haemocytes of mussel Mytilus galloprovincialis in the presence or the absence of micromolar concentrations of cadmium (Cd). Specifically, haemocytes exposed to different concentrations of TA (1, 10, 20, 40 and 60 μΜ) for 1 h, showed a significant reduction of their viability, only in concentrations higher than 40 μΜ. Furthermore, cells exposed to micromolar concentrations of Cd (50 and 100 μΜ), showed significantly increased levels of cell death, compared to those observed in control cells. In order to investigate the antioxidant or pro-oxidant ability of TA, haemocytes pre-treated for 15 min with different concentrations of TA were exposed to micromolar concentrations of the metal. According to the results, cells pre-treated with TA 1-40 μΜ, showed a significant attenuation of Cd induced effects, such as the production of superoxide (∙O2 −) and nitric oxides (NO), MDA content as well as cell death, compared to those occurred in the presence of the metal alone. On the contrary, in cells pre-treated with TA 60 μΜ before their exposure to the metal, TA seemed to act synergistically with the metal. The results of the present study could lead to the suggestion that TA in concentrations ranged within 1 and 40 μΜ could act as a major antioxidant factor, whereas in higher concentrations TA could cause oxidant effects, similar with those caused by well-known pro-oxidants, such as cadmium.

The aim of this thesis is to characterize metabolic changes in hemocytes during the immune response in D. melanogaster using in vivo markers as well as by measuring gene expression. The impact of the transcription factor HIF1 on the gene expression of glycolytic enzymes and its impact on the systemic metabolism was evaluated. The importance of HIF1 and LDH in the process of fighting against S. pneumoniae infection was tested as well.

Os invertebrados impedem o estabelecimento de uma infecção através de uma resposta imune eficiente. Esta resposta envolve reações celulares e humorais. Existem poucos trabalhos sobre a imunidade das aranhas. O principal objetivo deste estudo foi verificar o papel dos hemócitos e dos peptídeos antimicrobianos na resposta imune da aranha Acanthoscurria gomesiana. Inicialmente, a localização relativa da gomesina e da acanthoscurrina foi determinada, mostrando que 58% dos hemócitos armazenam os dois peptídeos antimicrobianos. Além disso, foi verificado que a gomesina é direcionada aos grânulos dos hemócitos da forma de pró-peptídeo. Observou-se ainda, que após um desafio, os hemócitos migram para o sítio de infecção, onde deve secretar componentes da cascata de coagulação e peptídeos antimicrobianos. Além disso, os resultados mostraram que a fagocitose não é o principal mecanismo ativado após uma infecção. Assim sendo, as principais respostas imunes da aranha são através da coagulação e secreção dos peptídeos antimicrobianos. / Invertebrates avoid the infection establishment through an efficient immune response. This response consists in cellular and humoral reactions. Few data are available concerning the spider\'s immunity. The main aim of this study was to determine the role of hemocytes and antimicrobial peptides on the immunity of the spider Acanthoscurria gomesiana. Initially, the localization of gomesin and acanthoscurrin was determined, showing that 58% of hemocytes store both antimicrobial peptides. Moreover, our results show that gomesin is addressed to the hemocyte granules as a pro-peptide. We also demonstrate, by in vivo and in vitro experiments, that hemocytes migrate to the site of microbial infection. Once at the site of infection, hemocytes might secrete components of coagulation cascade and antimicrobial peptides. Besides, our results suggest that phagocytosis is not the major defense mechanism activated after microbial challenge. Therefore the main reactions involved in the spider defense might be through the coagulation and antimicrobial peptides secretion.

Orientador: Eliana Maria Zanotti-Magalhães / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-05T18:40:54Z (GMT). No. of bitstreams: 1 Bruno_TreziaIedaBallerini_D.pdf: 6507064 bytes, checksum: 5515a1ea19ac4cfea55f25a3da2f2efa (MD5) Previous issue date: 2005 / Resumo: Experimentalmente, Biomphalaria glabrata pode ser utilizada como hospedeiro intermediário do nematódeo Angiostrongylus costaricensis, responsável pela angiostrongilíase abdominal. Esta zoonose, descrita no Brasil principalmente nos estados sulinos, pode acometer acidentalmente o homem, sendo que a infecção ocorre através da ingestão de moluscos parasitados ou água e alimentos contaminados com larvas de 3° estágio, eliminadas no muco dos moluscos hospedeiros. O objetivo deste trabalho foi estudar o desenvolvimento dos estágios larvais e o comportamento dos hemócitos na hemolinfa de B. glabrata infectada.Um total de 168 moluscos foi infectado com 120 larvas LI de A. costaricensis extraídas das fezes de camundongos Swiss albinos previamente infectados via oral sob tubagem esofágica com 6 larvas L3. Larvas de A. costaricensis foram recuperadas de 45 moluscos B. glabrata após 15, 22 e 29 dias de exposição ao parasita, através do método de Baermann, utilizando tecidos digeridos dos moluscos com solução de pepsina e ácido clorídrico. Constatou-se maior recuperação de larvas de A. costaricensis dos moluscos aos 29 dias de infecção. Para o estudo do desenvolvimento de Ao costaricensis, 60 moluscos infectados foram destinados a recuperação larval durante 30 dias consecutivos. Foi observada a mudança larval de LI para L2 aos 13 dias de infecção e L2 para L3 aos 18 dias de infecção. Hemolinfa de 45 moluscos infectados e não infectados com A. costaricensis foi coletada para verificação da resposta hemocitária durante 4 semanas. Os hemócitos foram distinguidos em hialinócitos e granulócitos. Enquanto nos moluscos não infectados predominaram os hialinócitos, naqueles infectados os granulócitos foram mais evidentes, principalmente entre o 18° ao 25° dia de infecção. Foi confirmada a ocorrência tanto da infecção percutânea como por via oral. Os locais mais parasitados foram: região cefalopodal, a preferida pelo nematódeo, seguida do intestino, rim e pulmão. Todas as larvas encontradas estavam viáveis e rodeadas por reação do tipo granulomatosa, independentes de sua localização / Abstract: Biomphalaria glabrata can be experimentally used as an intermediate host of the nematode Angiostrongylus costaricensis, responsible for abdominal angiostrongyliasis. This zoonosis, found in Brazil mainly in the southem states, can accidentally infect man through the ingestion of parasitized mollusks or contaminated water and food containing third-stage larvae, eliminated in the mucous secretion of the mollusks. The objective of this work was to study the development of larval stages and the behavior of hemocytes in the hemolymph of infected B. glabrata. A total of 168 mollusks were infected with 120 LI larvae of A. costaricensis, extracted ftom excrement of albino Swiss mice previously infected via the oral route by esophageal tube with 6 L3 larvae. The A. costaricensis larvae had been recovered from 45 B. glabrata mollusks at 15, 22 and 29 days after exposure to the parasite, by means of the method of Baermann, using molluscan tissues digested with pepsin and hydrochloric acid solution. A larger recovery of A. costaricensis larvae from the mollusk was found at 29 days after infection. For the study of the development of A. costaricensis, 60 infected mollusks were allocated for larval recovery during a period of 30 consecutive days. It was observed that there was a larval stage change, from L1 to L2, at the 13th day after infection and from L2 to L3 on the 18th day after infection.The hemolymph of 45 mollusks, both infected and not infected with A. costaricensis, was collected for verification of the hemocyte response during 4 weeks. The hemocytes were differentiated into hyalinocytes and granulocytes. While in the non infected mollusks the hyalinocytes had predominated, in those infected granulocytes were more evident, mainly between the 18th and the 25th day after infection. The occurence of infection, both via percutaneous and via oral routes, was confirmed. The most parasitized sites were the cephalopodan mass, preferred by the nematodes, folIowed by the intestines, kidneys and lungs. AlI the larvae found were viable and surrounded by reaction of the granulomatous type, independent of their situation / Doutorado / Mestre em Parasitologia

L’extension des activités humaines est responsable du rejet de molécules et de perturbations climatiques pouvant affecter la physiologie des organismes aquatiques. La moule zébrée possède des caractéristiques biologiques faisant d’elle une espèce intéressante en surveillance environnementale. Chez cet organisme, les hémocytes constituent une cible privilégiée pour la mise en place d’une approche multi-biomarqueurs. En effet, ces cellules à fonctionnalités multiples sont impliquées dans les grandes fonctions physiologiques de l’espèce et la régulation de l’homéostasie des individus. L’objectif de ce travail est de développer les outils analytiques permettant d’étudier les réponses hémocytaires de la moule zébrée. Les expérimentations menées ont permis de caractériser la structure des populations hémocytaires ainsi que leurs fonctionnalités propres en lien avec le processus de phagocytose. L’utilisation de ces biomarqueurs dans divers contextes indique une forte adaptabilité de l’espèce aux conditions environnementales. Les résultats montrent l’intérêt d’analyser les activités hémocytaires à l’échelle des sous populations comparativement à l’approche globale ne tenant pas compte de la diversité cellulaire. Il a été observé que certains facteurs comme le statut reproducteur ou l’espèce échantillonnée (D. polymorpha vs D. bugensis) constituent des facteurs de confusion importants. Il ressort également un positionnement fort du test de phagocytose en tant que marqueur de sensibilité aux contaminants. Ce travail constitue un ensemble de données voué à être utilisé dans des contextes multiples aussi bien en écotoxicologie qu’en écophysiologie. / Extension of human activities is responsible of molecule releases and climate changes that may affect physiology of aquatic organisms. The zebra mussel has biological traits making it an interesting species for environmental monitoring. In this organism, hemocyte cells constitute an interesting target to develop a multi-biomarker approach. These cells possess multiple functionalities and are involved in all major physiological functions of the species and in homeostasis regulation. The objective of this work was to develop analytical tools to study hemocyte responses of zebra mussels. Experiments allowed characterizing structure of hemocyte populations and their functionalities linked with phagocytosis process. Use of these biomarkers in various contexts indicated an important adaptation capacity of the species to environmental conditions. Results highlighted interest to analyze hemocyte activities at sub-population scale comparatively to global approach that does not consider hemocyte diversity. It was demonstrated that factor such as reproductive status or sampled species (D. polymorpha vs D. bugensis) constitute important confounding factors. Studies also demonstrated a strong positioning of phagocytosis assay as a sensitive marker to contaminants. This work constitutes a data set destined to be used in multiple contexts such as ecotoxicology or ecophysiology.

No description available.

Cell death shapes multicellular organism development and sustains tissue and organ homeostasis. Over the past decade we have begun to understand the breadth of physiological and biochemical diversity in cell death and clearance pathways, which play vital roles in organismal development and heath. While apoptosis and necrosis have been studied extensively across many model systems and contexts, the discovery of non-apoptotic paradigms of cell death and their roles in disease has greatly expanded the field. Collectively called Regulated Cell Death (RCD), these death pathways are regulated in a tissue and context-dependent manner (e.g. disease state). This dissertation is a culmination of multiple projects investigating cell death and clearance events spanning the ovary and the brain of the model organism, Drosophila melanogaster. We undertook the first multi-modal, high-throughput survey, involving single-cell RNA-seq, TRAP-seq, and proteomics, to compare two different archetypes of germline death in the fly egg chamber - apoptosis and phagoptosis. Our analysis identified several important candidates and pathways that are either unique to or shared between the germline death modalities and affecting oogenesis upon their disruption. We also observed that V-ATPases, proton pumps required for germline phagoptosis, are differentially localized throughout oogenesis, and we identified the specific subunits upregulated in phagoptosis. Furthermore, we identified a novel exon splicing event in the ‘a’ subunit isoform of V-ATPases that may facilitate its sub-cellular localization. Using a novel image analysis method involving image segmentation and spatial statistical inference, we determined that circulating immune cells agglomerate at specific niches within the abdomen, in response to egg chamber degeneration resulting from physiological stress of protein-deprivation. We then turned our focus to phagocytosis in the fly brain, which is essential for pruning synapses and for the removal of dying neurons and misfolded proteins. Disruptions to glial phagocytosis results in a range of age-dependent neurodegenerative phenotypes, primarily exemplified by vacuolization of brain tissue. Using a pre-trained deep-learning model to perform image segmentation and 3D reconstruction of vacuoles, we characterized the severity of neurodegeneration in brains lacking the phagocytic receptor Draper in glia and further demonstrated that this phenotype is attenuated by knockdown of the NF-κB transcription factor Relish in flies lacking glial Draper. Collectively, the methods and results described herein will have applications beyond the Drosophila model and the field of cell death, with important implications in understanding fertility and the underpinnings of cognitive disorders.