Tesi: "Fish cell line"

1

Dumont, Sarah. "Caractéristiques cliniques, moléculaires et prise en charge des Rhabdomyosarcomes de l'adulte et identification d'une polythérapie ciblée in vitro". Thesis, Lyon 1, 2013. http://www.theses.fr/2013LYO10311/document.

Testo completo

Gli stili APA, Harvard, Vancouver, ISO e altri

Abstract (sommario):

Le rhabdomyosarcome de l'adulte est une tumeur rare au pronostic. Le présent travail propose d'étudier les caractéristiques cliniques et moléculaires et la prise en charge des adolescents et adultes atteints de rhabdomyosarcome ainsi que la possibilité de combinaison de thérapie ciblées sur lignées cellulaires in vitro. Nous avons anamysé rétrospectivement 239 patients âgés de 10 ans ou plus, atteints de rhabdomyosarcome au MD Anderson Cancer Center entre 1957 et 2003 et leur statut fusionnel pour PAX-FOXO1 par hybridation in situ en fluorescence. Trois lignées cellulaire de sarcome à petites cellules ont été soumises à des combinaisons de thérapies ciblées avec analyse de la viabilité. Les patients de plus de 50 ans avaient une survie globale à 5 ans de 13 % (médiane de survi à 1.7 ans) en dépit d'une maladie localisée. Approximativement 13 % des patients métastasiques de moins de 50 ans ont eu une survie prolongée de plus de 15 ans. L'utilisation d'une stratégie thérapeutique triple, intégrant chirurgie, chimiothérapie et radiothérapie était signifcativement associée à une survie prolongée. Auniveau molécualire, la présence du transcrit de fusio PAX3/7-FOXO1 était significativement liée à un risque accru de maladie métastatique. L'étude in vitro de thérapies ciblées a permis d'identifier la combinaison du vorinostat plus le 17DMAG associée à la doxorubicine comme ayant une meilleure efficacité. La prise en charge du rhabdomyosarcome de l'adolescent et de l'adulte semble souffrir d'une approche moins agressive comparée au rhabdomyosarcome pédiatrique. De plus, des combianaisons de thérapies ciblées peuvent être intégrées aux protocoles de chimiothérapies standards
Rhabdomyosarcoma is a rare entity adult patient with unfavourable outcome. This work describes the clinical and molecular specificities of adolescent and adult type of rhabdomyosarcoma and investigates the optimal integration of targetd therapy combinations on small cell sarcoma cell lines in vitro. We retrospectively analyzed 239 patients, 10 years of age and greater, diagonsed withrhabdomyosarcoma at MD Anderson Cancer Center from 1957 trough 2003 and their PAX-FOXO1 fusion gene status by fluorescence in situ hybridization on tissues microarray. Three samll cell sarcoma cell lines were exposed to targetd agent combinations. PAtient with metastatic rhabdomyosarcoma were found to have a 18 % survival rate at 5 years from diagnosis with an 12 %survival past 15 years. This outcome was even poorer for patients over 50 of age, even with localized disease. Younger patients were more likely to receive multidisciplinary therapy than their older counterparts. The presence of PAX-FOXO1 tranlocation was significantly associated with a higher frequency of metastatic disease. The four agents with the exception of abacavir synergized two by two with each other in vitro but the triple combinations did not perform beter than the bitherapies. The dual therapies vorinostat 5HDAC inhibitor) plus 17-DMAG (Hsp90 inhibitor) added with doxorubicin achvied better results than dual or triple therapies. Adult patient with rhabdomyosarcoma present similar molecular and clinical characteristics compared pediatric patients but outcome decrease with age partly du to a less multimodal management. Moreover targeted combinations should be integrated to chemotherapy backbone

2

Nehls, Sebastian. "Anwendung des Comet Assay (Einzelzell-Gelelektrophorese) an Zellen von Fischen zum Nachweis gentoxischer Wirkungen im aquatischen Biomonitoring". Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2013. http://dx.doi.org/10.18452/16830.

Testo completo

Gli stili APA, Harvard, Vancouver, ISO e altri

Abstract (sommario):

Gewässer sind Lebensgrundlage, jedoch gleichzeitig Schadstoffsenken für eine Vielzahl von Kontaminanten. Biologische Wirkungstests und das Biomonitoring aquatischer Proben sind daher besonders wichtig, um Umwelt-Gefahrenpotenziale erkennen zu können. Der "Comet Assay" (Einzelzell-Gelelektrophorese) ist ein Indikator von DNA-Strangbrüchen und wurde hier als Test auf gentoxische Wirkungen erprobt und angewandt. Mit bekannten, gentoxischen Substanzen wurden Nachweisgrenzen und Dosis-Wirkungs-Beziehungen für die Zelllinien RTG-2 und RTL-W1 (aus der Regenbogenforelle, Oncorhynchus mykiss) in vitro ermittelt und methodische Parameter an die Zellen angepasst. Der Test reagierte sehr sensitiv auf 4-Nitrochinolin-1-oxid. Die Substanz war daher geeignet, um in weiteren Versuchen als Positivkontrolle zu dienen. Zur Bewertung der Messdaten wurde ein geeignetes statistisches Verfahren gefunden, das auch historische Kontrollen mit einbezog. Der zeitliche Verlauf der DNA-Schädigung des Testsystems mit RTG-2-Zellen wurde ermittelt, und durch Inhibition der DNA-Reparatur mit Aphidicolin wurden Zusammenhänge zwischen der Entstehung von DNA-Strangbrüchen, der DNA-Reparaturkapazität sowie der Metabolisierungskapazität untersucht. In einer zweiten Phase wurden unbehandelte Wasserproben aus Rhein, Elbe sowie weitere Oberflächenwasserproben mit dem Comet Assay an RTG-2-Zellen getestet. Bei 15 von 49 Proben zeigten sich gentoxische Effekte. In einer dritten Phase wurden Erythrozyten von freilebenden Döbeln, Leuciscus cephalus, aus der Mosel mit dem Comet Assay untersucht. Die Fische von drei Messstellen zeigten erhöhte Werte von DNA-Schädigungen, gegenüber einer vierten, stromabwärts gelegenen Messstation. Korrelationen mit den Ergebnissen zusätzlicher Biomarker ergaben sich nur teilweise. Chemische Analysen von Wasser- oder Gewebeproben ließen keine Rückschlüsse auf verursachende Kontaminanten zu - gerade dies unterstreicht jedoch die Wichtigkeit biologischer Tests bei komplexen Proben.
Bodies of Water are both vital resources and pollutant sinks for a multitude of contaminants. Therefore, biological effect tests and biomonitoring of aquatic samples are of particular importance to detect potential environmental hazards. The "comet assay" (single cell gel electrophoresis) is an indicator for DNA strand breaks and was explored and applied as a genotoxicity test in the present study. Known genotoxic substances were used to determine the detection limits and dose-response relationships for the cell lines RTG-2 and RTL-W1 (from rainbow trout, Oncorhynchus mykiss) in vitro, and to adapt methodological parameters to the cells. The test was very sensitive to 4-Nitroquinoline-1-oxide. This substance was therefore well-suited to serve as positive control in further experiments. In order to evaluate the measurement data, an appropriate statistical procedure was developed, which also took "historical" controls into account. The time course of DNA damage in the test system using RTG-2 cells was determined, and relationships between the origin of DNA strand breaks, DNA repair capacity and the metabolizing capacity of the cells was investigated by means of inhibition of DNA repair with Aphidicoline. In the second stage, native water samples from the rivers Rhine and Elbe and further surface waters were tested with the comet assay, using RTG-2 cells. 15 out of 49 samples showed genotoxic effects. In a third stage, erythrocytes of feral chub, Leuciscus cephalus, from the Moselle river were examined with the comet assay. The fish from three measuring stations showed elevated values of DNA damage compared to fish sampled from a downstream station. There were only partly correlations with the results from additional biomarkers. Chemical analyses of water and tissue samples did not permit conclusions on effect-causing substances.However, this emphasizes the importance of biological tests in dealing with complex environmental samples.

3

DeWitte-Orr, Stephanie. "A study of innate antiviral mechanisms using fish cell lines". Thesis, University of Waterloo, 2006. http://hdl.handle.net/10012/1272.

Testo completo

Gli stili APA, Harvard, Vancouver, ISO e altri

Abstract (sommario):

Understanding basic antiviral mechanisms in vertebrates is essential for developing methods to enhance antiviral responses and promote human and animal health. In fish these antiviral mechanisms are poorly understood, but are important to understand because of the devastating impact of viral diseases on aquaculture. Therefore, the antiviral responses of a rainbow trout macrophage-like cell line, RTS11, and two non-immune cell lines, the rainbow trout fibroblast RTG-2 and Chinook salmon embryo CHSE-214 were studied. Three antiviral responses were first characterized using the viral mimic, synthetic double-stranded RNA (poly IC), and then their induction was investigated using Chum salmon reovirus (CSV). The responses were: 1) apoptosis, which is programmed cell death and a primitive antiviral defense; 2) homotypic aggregation (HA), which is clustering of like immune cells; and 3) expression of Mxs, which are antiviral proteins belonging to GTPase super-family. Some of these antiviral mechanisms were investigated using a novel continuous cell line, PBLE, developed from a peripheral blood leukocyte preparation of the American eel, Anguilla rostrata.

RTS11 was exceptionally susceptible to apoptosis. The cells died at lower concentrations of poly IC and other agents, including the translation inhibitor, cycloheximide (CHX), and fungal metabolite, gliotoxin. Death was predominantly by apoptosis, as judged by DNA ladders, nuclear fragmentation, and protection by caspase inhibitors. By contrast, the other two cell lines died most commonly by necrosis, when death did occur. Co-treating RTS11 with CHX greatly sensitized the cells to poly IC. Based on the protection afforded by inhibitors of dsRNA-dependent protein kinase (PKR), RTS11 apoptosis induced by poly IC with CHX co-treatment but not gliotoxin was mediated by PKR. As macrophages are likely among the first cells to contact viruses during an infection in vivo and are mobile, the sensitivity of RTS11 to dsRNA killing could reflect a protective mechanism by which virus spread is limited by the early death of these first responders.

HA of RTS11 was induced by poly IC. HA required divalent cations and was blocked by CHX and by PKR inhibitors. This suggested that HA induction was PKR-mediated and involved the synthesis of new cell surface molecule(s), possibly galectins. As an antiviral mechanism, HA induction by dsRNA could be interpreted as an initial protective response, allowing cell localization at the site of infection, but once translation becomes inhibited, apoptosis ensues.

Mx was induced by poly IC in RTS11 and RTG-2 as judged by RT-PCR. Western blotting revealed constitutive Mx expression more consistantly in RTS11, but induction by poly IC in both cell lines. Medium conditioned by cells previously exposed to poly IC and assumed to contain interferon also induced Mx transcripts in RTS11 but not RTG-2. In RTS11, poly IC activated PKR activity, and PKR inhibitors blocked Mx induction, which is the first demonstration of PKR mediating Mx expression.

The dsRNA virus, CSV, also induced apoptosis, HA, and Mx expression, but in some cases contrasting with poly IC experiments. CSV induced apoptosis in RTG-2 and CHSE-214 but not in RTS11, and HA induction by CSV in RTS11 was not dependent on PKR. Mx induction was sustained in RTG-2 and transitory in RTS11; however, both cell lines supported CSV replication.

The novel cell line, PBLE, was also characterized in this study. PBLE was derived from an adherent culture of peripheral blood leukocytes from the American eel, Anguilla rostrata. PBLE were found to grow over a wide range of temperatures and fetal bovine serum (FBS) concentrations. This cell line was able to undergo apoptosis in response to gliotoxin. PBLE was also susceptible to a number of viruses, including CSV; however, CSV infection did not lead to apoptosis.

This study suggests that antiviral responses are likely numerous and overlapping and depend on cell type and virus. Understanding them should lead to novel methods for protecting fish from viral diseases. More specifically, using cell lines such as PBLE may aid in the understanding of species specific and perhaps even cell type specific antiviral mechanisms.

4

John, K. Riji. "Characterization of reovirus-like agents associated with snakehead fish and cell culture". Thesis, University of Stirling, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.244645.

Testo completo

Gli stili APA, Harvard, Vancouver, ISO e altri

5

Gignac, Sarah Jane. "Characterization of Fundulus heteroclitus embryonic cell lines and their applications to fish health". Thesis, University of British Columbia, 2014. http://hdl.handle.net/2429/46430.

Testo completo

Gli stili APA, Harvard, Vancouver, ISO e altri

Abstract (sommario):

Common killifish, or mummichogs (Fundulus heteroclitus), are a species of estuarine teleost that are widely used in comparative physiology, toxicology and embryology. Their ability to withstand extreme environmental conditions, widespread distribution, and relatively sedentary nature, makes them ideal as sentinel species of estuarine health. However, the lack of cell lines derived from F. heteroclitus places limitations on the utility of this species in environmental research. In contrast, cell cultures derived from other model organisms have assisted and facilitated our understanding of the effects that environmental contaminants have on organisms in vitro. The development and use of novel F. heteroclitus cell lines for toxicological and parasitological applications is reported here. Continuous proliferating cells were derived from pre-hatch embryos of killifish and have been maintained for 3 years. Three stable cell lines were obtained from the head and body tissues of F. heteroclitus; these stable cell lines have been dubbed KilliFish Embryo 1, 3, and 5 (KFE-1, KFE-3, KFE-5). All three cell lines have been characterized for origin and functionality, as well as for applications in toxicology, studying effects of model chemical pollutants, and in parasitology to evaluate a cod-infecting microsporidia that has been an emerging pathogen of concern, for their ability to infect and grow in cell lines derived from sentinel species. KFE-1 has characteristics of neuroepithelial cells, whereas KFE-3 are possibly liver derived cells, and KFE-5 are distinctly myogenic, as this line has cells that appear to be striated muscle cells. Like intact F. heteroclitus, these cell lines can withstand a wide temperature range from 4°C to 37°C. Mechanisms of thermotolerance and ability to withstand salinity and hypoxia as well as chemical toxicity tolerance could be readily studied with these new cell lines.

6

Fleurbaix, Emmanuel. "Évaluation écotoxicologique des éléments terres-rares : approches cellulaires chez différentes espèces aquatiques". Electronic Thesis or Diss., Université de Lorraine, 2021. http://www.theses.fr/2021LORR0324.

Testo completo

Gli stili APA, Harvard, Vancouver, ISO e altri

Abstract (sommario):

Depuis 30 ans, l’utilisation croissante des Lanthanides dans les nouvelles technologies a entraîné des rejets importants de ces métaux vers les écosystèmes aquatiques. Dans une politique mondiale de développement durable visant à préserver la qualité des écosystèmes, la question de l’impact des Lanthanides sur les organismes aquatiques s’est naturellement posée. Néanmoins, les études restent peu nombreuses et aucun consensus ne subsiste concernant la toxicité des Lanthanides. Dans ce contexte, nous avons étudié la toxicité cellulaire des Lanthanides individuellement et en mélanges. Les effets toxiques ont été mis en évidence en mesurant la viabilité de cellules fibroblastiques (ZF4 ; ATCC®, CRL-2050™) et hépatiques (ZFL ; ATCC®, CRL-2643™) de poisson zèbre (Danio rerio), de cellules branchiales (RTgill-W1 ; ATCC®, CRL-2523™) de la truite arc-en-ciel (Oncorynchus mykiss), et des cellules primaires de glandes digestives de corbicule (Corbicula fluminea) exposées à ces métaux. Les résultats ont montré que les Lanthanides sont responsables d’effets toxiques directs sur nos modèles cellulaires. Concernant la toxicité des Lanthanides en mélanges, des effets synergiques ont été observés sur les 3 lignées cellulaires de poissons. Nous nous sommes également intéressés aux mécanismes de détoxification des Lanthanides dans les cellules ZF4 de Danio rerio. Nous avons décidé d’étudier ces acteurs en raison de leurs rôles respectifs dans les phases II et III de la détoxification cellulaire de métaux chez les bivalves et les poissons. Pour cela, la viabilité des cellules ZF4 a été mesurée après des expositions aux Lanthanides en présence d’inhibiteurs spécifiques des glutathion-S-transférases (acide éthacrynique) et des protéines MRP (MK571 et probénécide). Les résultats ont montré que les protéines MRP sensibles au MK571 jouent un rôle dans la détoxification des Lanthanides dans les cellules ZF4. Globalement, les résultats obtenus pour cette recherche ont confirmé que les effets toxiques des Lanthanides à l’échelle cellulaire sont pertinents pour prédire les effets in vivo, dans le cadre d’une évaluation de la toxicité de ces métaux
Since 30 years ago, the growing use of Lanthanides in new technologies has contributed to important releases of these metals into aquatic ecosystems. In a global sustainable development policy aimed at preserving the quality of ecosystems, the impact of Lanthanides on aquatic organisms has naturally been questioned. However, studies on the aquatic ecotoxicology of Lanthanides are incomplete, and no consensus is established yet. In this context, we studied the cellular toxicity of Lanthanides individually and in mixtures. To determine these toxic effects, cell viability was measured on Danio rerio fibroblast-like cells (ZF4; ATCC®, CRL-2050™), Danio rerio hepatic cells (ZFL; ATCC®, CRL-2643™), Oncorhynchus mykiss epithelial cells (RTgill-W1; ATCC®, CRL-2523™), and primary culture of Corbicula fluminea digestive glands exposed to Lanthanides. Direct toxicity of Lanthanides has been observed on all cellular models. Concerning the toxicity of Lanthanides in mixtures, synergistic effects have been underlined on the three fish cell lines. In this research, we focused on the mechanisms of the detoxification of Lanthanides in the case of ZF4 cells from Danio rerio. The effects of Lanthanides were assessed in the presence of specific inhibitors of glutathione-S-transferases (ethacrynic acid) and MRP-like (MK571 and probenecid), by cell viability measurements. We decided to study these actors of the cellular detoxification due to their respective roles in phases II and III of the cellular detoxification of metals in fishes and bivalves. Regarding the results, MRP-like proteins are effectively involved in the detoxification of Lanthanides in ZF4 cells. Overall, our results highlighted the relevance of the toxic effects of Lanthanides at the cellular level for the risk assessment of these metals

7

Wiersinga-Post, Jenny Esther Clasine. "Mechanophysiology of cupulae and hair cells in the lateral line of fish and pitch perception of complex sounds in humans". [S.l. : [Groningen] : s.n.] ; [University Library Groningen] [Host], 1997. http://irs.ub.rug.nl/ppn/164044949.

Testo completo

Gli stili APA, Harvard, Vancouver, ISO e altri

8

Flemming, Leonard. "Comparative proteomic and genomic analysis of Flavobacterium johnsoniae-like biofilm, planktonic and agar surface-associated cells". Stellenbosch : University of Stellenbosch, 2010. http://hdl.handle.net/10019.1/4020.

Testo completo

Gli stili APA, Harvard, Vancouver, ISO e altri

Abstract (sommario):

Thesis (PhD(Microbiology))--University of Stellenbosch, 2010.
ENGLISH ABSTRACT: Pathogenic Flavobacterium spp. cause serious disease outbreaks in a variety of farmed fish, which lead to large economic losses in the aquaculture industry on an annual basis. The ability of Flavobacterium johnsoniae-like isolates to grow as surface-associated communities (biofilms) in aquaculture systems poses a threat to fish health over extended periods of time. The biofilmforming ability of 28 F. johnsoniae-like isolates obtained from diseased fish were correlated with their chitin-degrading abilities and extracellular carbohydrate complexes (ECC) and their pulsed-field gel electrophoresis (PFGE) genotypes. Physiological changes in the proteome of 5 day planktonic, biofilm and agar surface-associated cultures of F. johnsoniae-like isolates YO12 and YO64 were analyzed by two-dimensional (2-D) gel electrophoresis and 17 differentially expressed and 14 uniquely expressed proteins were identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Thirty-two differentially expressed genes in 5 day biofilm and agar surface-associated cultures of F. johnsoniae-like isolates YO12 and YO64 were identified using suppression subtractive hybridization (SSH). Significant negative correlations were observed between the chitin-degrading abilities and ECC and the biofilmforming capacity of 24 h biofilm cultures of F. johnsoniae-like isolates. Genetic heterogeneity was displayed by the F. johnsoniae-like isolates following PFGE. A significant positive correlation was observed between PFGE types and fish host species. Differentially and uniquely expressed proteins identified in planktonic, biofilm and agar surface-associated phases by 2-D/MS as well as differentially expressed genes identified in the biofilm and agar surface-associated phases by SSH were categorized as being involved in adaptation/protection, metabolic processes, membrane/transport/ motility and transcription/ translation. As far as we know, this is the first report on the characterization of differentially expressed genes and gene products of F. johnsoniae-like isolates obtained from diseased fish in South Africa.
AFRIKAANSE OPSOMMING: Patogene Flavobacterium spp. veroorsaak ernstige infeksie uitbrake in ’n verskeidenheid gekweekte vissoorte, wat jaarliks tot groot ekonomiese verliese in die akwakultuur bedryf lei. Die vermoë van Flavobacterium johnsoniae-tipe isolate om as oppervlak-gehegde gemeenskappe (biofilms) in akwakultuur sisteme te groei bedreig visgesondheid oor verlengde periodes. Die vermoë van 28 F. johnsoniae-tipe isolate om biofilms te vorm is vergelyk met hul vermoë om chitien te degradeer, die profiel van hul ekstrasellulêre koolhidraat komplekse (EKK) en bandpatrone verkry met puls-veld jel elektroforese (PVJE). Fisiologiese veranderinge in die proteoom van 5-dagoue planktoniese-, biofilm- en agar oppervlak-geassosieerde kulture van F. johnsoniae-tipe isolate YO12 en YO64 is met twee-dimensionele (2-D) jel elektroforese geanaliseer. Sewentien differensieël uitgedrukte en 14 uniek uitgedrukte proteïene is deur middel van matriks-geassisteerde laser desorpsie ioniserings-tyd van vlug-massa spektrometrie (MGLDI-TVV MS) geïdentifiseer. Twee-en-dertig differensieël uitgedrukte gene in 5-dag-oue biofilm- en agar oppervlak-geassosieerde kulture van F. johnsoniae-tipe isolate YO12 en YO64 was deur middel van suppressie afgetrokke hibridisasie (SAH) geïdentifiseer. Beduidende negatiewe korrelasies is tussen die chitin-degraderings vermoë en EKK en die biofilm-vormings kapasiteit van 24-uur-oue biofilm kulture van F. johnsoniae-tipe isolate waargeneem. Resultate verkry met PVJE het die heterogene samestelling van F. johnsoniae-tipe isolate uitgewys. ‘n Beduidende positiewe korrelasie is tussen PVJE groeperings en vis gasheer spesie waargeneem. Differensieël en uniek uitgedrukte gene geidentifiseer in die planktoniese-, biofilm- en agar oppervlak-geassosieerde fases is deur middel van 2-D/MS asook differensieël uitgedrukte gene geïdentifiseer in die biofilm en agar oppervlakgeassosieerde fases deur middel van SAH was as betrokke by aanpassing/beskerming, metaboliese prosesse, membraan/vervoer/ beweeglikheid en transkripsie/translasie gekategoriseer. Sover bekend is hierdie die eerste beskrywing van differensieël uitgedrukte gene en geenprodukte van F. johnsoniae-tipe isolate afkomstig van geinfekteerde vis in Suid Afrika.

9

Privitera, Giovanna. "Studio di nuovi approcci farmacologici in grado di inibire l'attivazione dei recettori del fattore di crescita epidermico (EGF) in linee cellulari di carcinoma polmonare non a piccole cellule (NSCLC)". Doctoral thesis, Università di Catania, 2013. http://hdl.handle.net/10761/1436.

Testo completo

Gli stili APA, Harvard, Vancouver, ISO e altri

Abstract (sommario):

Il carcinoma del polmone rappresenta attualmente la neoplasia più frequentemente diagnosticata e costituisce la principale causa di morte per tumore solido al mondo. E una malattia multifattoriale che riconosce nella cancerogenesi cause ambientali e cause genetiche. L istotipo più frequente è il carcinoma non a piccole cellule (NSCLC) che rappresenta circa il 75-80% delle diagnosi. Il principale approccio terapeutico consiste nella resezione chirurgica (eventualmente preceduta e/o seguita da chemioterapia) limitata però ai soli stadi iniziali. Studi precedenti hanno dimostrato che l iper-espressione o l attivazione costitutiva dei recettori del fattore di crescita epidermico (EGF) è coinvolta nella crescita dei carcinomi umani, incluso quello polmonare. In questo lavoro sperimentale è stato studiato l effetto del blocco di due recettori dell EGF, EGFR e HER-2, sulla proliferazione delle cellule A549 e NCI-H226 di carcinoma polmonare. Sono stati utilizzati, a tale scopo, cetuximab, anticorpo monoclonale diretto contro l EGFR e trastuzumab, anticorpo monoclonale diretto contro l HER-2, a diverse concentrazioni per 72 h. Mediante il saggio di proliferazione MTT (3,(4,5-dimethylthiazol-2)2,5 difeniltetrazolium bromide) è stato osservato il 91,4% di inibizione della proliferazione delle cellule A549 trattate con una combinazione di cetuximab e trastuzumab alla concentrazione di 40 ug/ml, mentre le cellule NCI-H226 hanno mostrato una resistenza allo stesso trattamento combinato. Sono state successivamente valutate le eventuali variazioni di espressione dei livelli di mRNA dei recettori EGFR e HER-2 di entrambe le linee cellulari. Nelle cellule A549 è stato osservato un significativo incremento dei livelli di mRNA di EGFR dopo 30 h dal trattamento farmacologico, spiegabile considerando il meccanismo di azione del cetuximab, che determina internalizzazione e degradazione del recettore in tempi brevi. A questa segue quindi nuova sintesi di proteine recettoriali. Questi livelli di mRNA diminuiscono dopo 48 h e 72 h dal trattamento per avvicinarsi poi ai valori normali. Un comportamento analogo è stato osservato nei livelli di mRNA del recettore HER-2, in cui l internalizzazione del recettore, conseguente all interazione con il trastuzumab, determina già a 18 h di trattamento un incremento dell espressione di tale recettore. Le cellule NCI-H226 hanno mostrato invece un aumento di circa 3,5 volte dei livelli di mRNA dell EGFR dopo 48 h dal trattamento e il mantenimento di alti livelli anche dopo 72 h. Il recettore HER-2 non sembra essere invece influenzato dal trattamento farmacologico e, almeno nei primi tempi della somministrazione, i livelli di mRNA non vengono alterati. Solo dopo 72 h di trattamento è stato osservato un aumento di tali livelli, che vengono circa raddoppiati, ad indicare un possibile coinvolgimento tardivo di tale recettore nella risposta al trattamento combinato. L iniziale scarso coinvolgimento del recettore HER-2 potrebbe quindi essere responsabile della resistenza delle cellule NCI-H226. E pertanto nostro obiettivo futuro prolungare il tempo di trattamento delle cellule NCI-H226 con cetuximab e trastuzumab allo scopo di valutare un effetto antiproliferativo più tardivo. La differenza di sensibilità al cetuximab e al trastuzumab, nelle due linee cellulari prese in esame, non dipende invece dal numero di copie del gene, in quanto, mediante la tecnica di ibridazione in situ fluorescente (FISH), abbiamo osservato lo stesso incremento del numero di copie dei geni EGFR e HER-2 e quindi una condizione di polisomia, in entrambe le linee cellulari. La strategia di agire su entrambi i recettori potrebbe quindi migliorare il trattamento del carcinoma polmonare, aumentare la sopravvivenza e ridurre gli effetti collaterali della terapia, garantendo così al paziente un sensibile miglioramento della qualità di vita.

10

Kienzler, Aude. "Intérêt des lignées cellulaires de poisson en écotoxicologie pour l'étude de nouveaux biomarqueurs de génotoxicité". Phd thesis, INSA de Lyon, 2013. http://tel.archives-ouvertes.fr/tel-00952888.

Testo completo

Gli stili APA, Harvard, Vancouver, ISO e altri

Abstract (sommario):

Un contexte réglementaire de plus en plus strict en évaluation du risque écotoxicologique des milieux aquatiques exige de renforcer les outils d'évaluation. A ce titre, l'étude des biomarqueurs de génotoxicité doit être privilégiée, compte tenu du rôle central de l'ADN dans le fonctionnement du vivant. L'exposition à des agents génotoxiques peut générer des dommages par interaction directe avec l'ADN, mais aussi indirectement, en modulant l'efficacité des mécanismes de réparation de l'ADN, ou la régulation épigénétique de l'expression des gènes. Aujourd'hui, la plupart des biomarqueurs de génotoxicité visent les dommages primaires à l'ADN ou la mutagènicité mais les effets indirects sur sa fonctionnalité sont encore peu étudiés. Dans ce contexte, à l'issue d'une analyse bibliographique comprenant la rédaction d'une revue sur les mécanismes de réparation des dommages à l'ADN chez le poisson, ce travail visait au développement méthodologique de plusieurs biomarqueurs de génotoxicité à l'aide de trois lignées cellulaires pisciaires (RTL-W1, RTGill-W1 et PLHC-1). Pour ce faire, l'essai des comètes en conditions alcalines a été décliné sous plusieurs versions dans l'objectif d'utiliser une technique de base unique permettant la mesure complémentaire de plusieurs biomarqueurs de génotoxicité : les dommages primaires à l'ADN, les activités de réparation et le niveau de méthylation des cytosines du génome. Les résultats soulignent l'intérêt des trois lignées en évaluation de la génotoxicité 1) pour détecter in vitro de manière sensible des atteintes primaires à l'ADN de natures variées à de faibles concentrations grâce à un essai des comètes modifié par une étape de digestion enzymatique avec une glycosylase (Fpg), 2) pour évaluer l'influence des contaminants sur l'activité de réparation par excision de bases (BER) via la mesure de la capacité d'incision d'un ADN substrat porteur de lésions de type 8-oxoGua par des extraits cellulaires (essai BERc). Le niveau de méthylation des cytosines (5-meCyt) des lignées RTgill-W1et RTL-W1 a été mesuré par HPLC-MS-MS, leur valeur élevée permet d'envisager le paramètre méthylation comme biomarqueur potentiel. Ce volet nécessitera cependant des étapes de validations ultérieures car il n'a pas été techniquement possible de mettre au point un essai des comètes modifié pour la mesure du niveau de méthylation de l'ADN. Plusieurs activités de réparation des lignées RTgill-W1 et RTL-W1 ont été caractérisées et révèlent de bonnes aptitudes de réparation de type " Base Excision Repair " (BER) et " Photo Enzymatic Repair " (PER) et une plus faible capacité au " Nucleotide Excision Repair " (NER), soit un profil proche de celui décrit in vivo et sans différence marquée entre les deux lignées. Les biomarqueurs développés sur les lignées cellulaires de poisson au cours de ce travail ont également été appliqués à la mesure des effets génotoxiques d'effluents issus du lessivage de revêtements routiers.

11

Komegae, Evilin Naname. "Papel dos receptores inatos TLR na formação de memória humoral e linfócitos B de longa vida: ação das proteases natterinas, toxinas majoritárias do veneno de Thalassophryne nattereri". Universidade de São Paulo, 2010. http://www.teses.usp.br/teses/disponiveis/42/42133/tde-01102010-120643/.

Testo completo

Gli stili APA, Harvard, Vancouver, ISO e altri

Abstract (sommario):

A contribuição de células B para a memória imunológica se dá por duas distintas populações: células B de memória e células produtoras de anticorpos de longa vida (ASC). A inter-relação entre estas células bem como os mecanismos envolvidos para a manutenção destas tem sido pouco entendida. O veneno de Thalassophryne nattereri tem se mostrado capaz de induzir uma intensa resposta imune de memória. Nós avaliamos o efeito das Natterinas, que são as toxinas majoritárias e inéditas, na indução e manutenção da resposta imune de memória de células B. Este estudo, além de permitir um maior esclarecimento da resposta humoral de memória induzida pelo veneno do peixe T. nattereri, permitiu o estudo da complexa organização do compartimento de células B de memória e ASCs. Também evidenciamos a importância da atividade proteásica para a manutenção da cronicidade de resposta de células B no peritônio, no baço e na medula, como verificamos que a ativação de receptores inatos como osTLRs é decisiva para a geração e manutenção de ASCs B220pos/neg em resposta às Natterinas, dependentes das vias de sinalização MyD88 ou TRIF. Estas sinalizações controlam a magnitude, a qualidade e a longa duração da resposta humoral de memória.
The contribution of B cells for the immunological memory feels for two different populations: memory B cells and long-lived antibodies secreting cells (ASC). The interrelation among these cells as well as the mechanisms involved for the maintenance of these it has been little understood. The venom of Thalassophryne nattereri possesses the ability to induce an intense memory immune response. We evaluated the effect of Natterins that are majority toxins in the venom, in the induction and maintenance of the immune memory response of cells B. The study, besides allowing a larger explanation of the humoral memory response induced by the venom of the fish, it allowed the understanding of the complex organization of the memory B cells compartment, mainly of the subtype of long-lived cells (ASC). Also, we showed the importance of the protease activity of Natterins in the maintenance of the chronic B cell responses in the three analyzed compartments. We verify that the activation of Toll like receptors is decisive for the generation and maintenance of ASCs B220pos/neg in response to Natterins, dependent on the MyD88 or TRIF signaling that control the quality and the duration of the humoral memory response.

12

Hovnanian, Jessica. "Méthode de frontières immergées pour la mécanique des fluides : application à la simulation de la nage". Phd thesis, Université Sciences et Technologies - Bordeaux I, 2012. http://tel.archives-ouvertes.fr/tel-00835013.

Testo completo

Gli stili APA, Harvard, Vancouver, ISO e altri

Abstract (sommario):

Nous nous interessons à la modélisation des interactions fluide-structure entre un fluide visqueux incompressible et une structure pouvant être déformable. Apres une approche des méthodes de type frontière immergée existantes, nous présentons une nouvelle approche : la méthode IPC (Image Point Correction) que nous validons ensuite sur différents cas tests. Puis, nous l'appliquons à la simulation 2D puis 3D de la nage d'un poisson grâce à une reconstruction utilisant l'outil du squelette.

13

Devès, Mathilde. "Arrêt de la prolifération cellulaire pendant le développement embryonnaire : étude transcriptionnelle de gènes suppresseurs de tumeurs au cours de la croissance du système nerveux central chez le poisson médaka Oryzias latipes". Phd thesis, Université Paris Sud - Paris XI, 2012. http://tel.archives-ouvertes.fr/tel-00879146.

Testo completo

Gli stili APA, Harvard, Vancouver, ISO e altri

Abstract (sommario):

Comment la taille d'un organisme est-elle régulée au cours du développement embryonnaire ? Quels sont les mécanismes génétiques à l'origine de l'arrêt de la prolifération pendant la croissance d'un organisme pluricellulaire ? Afin d'identifier des acteurs de la sortie du cycle cellulaire au cours du développement, mon travail s'est orienté sur l'étude de gènes suppresseurs de tumeurs pendant la croissance du toit optique (TO) du médaka Oryzias latipes. Le TO, structure dorsale du cerveau moyen des Vertébrés, est un modèle particulièrement adapté à l'étude de la régulation de la prolifération. Trois zones de la marge vers le centre du TO sont discernables : une zone périphérique de prolifération, une zone intermédiaire de cellules sortant du cycle cellulaire et une zone centrale de cellules différenciées. Un crible d'expression par hybridation In Situ a été réalisé et a permis d'identifier 28 gènes exprimés dans le TO, suggérant leur implication dans le contrôle de la sortie du cycle cellulaire au cours du développement. Dans le but de caractériser in vivo la fonction de gènes issus de ce crible, le gène BTG1 (B-cell Translocation Gene 1) et les membres de sa famille, ont été étudiés au cours du développement du médaka. J'ai mené des expériences fonctionnelles sur BTG1, permettant de mettre en évidence son rôle central pour la morphogenèse du système nerveux central. De plus, une autre partie de mon travail s'est penchée sur l'étude de l'expression des membres de la voie de signalisation Hippo, bien connue et caractérisée chez la drosophile et les Mammifères pour son rôle dans le contrôle de la taille des organes via une régulation de l'arrêt de la prolifération. A l'issu de notre travail, une fonction de la voie de signalisation Hippo dans la formation du TO et des somites a pu être mise en évidence au cours du développement du médaka.

14

Pracucci, Enrico. "Unraveling alterations of excitation/inhibition balance in in vivo models of epilepsy and genetic autism". Doctoral thesis, Scuola Normale Superiore, 2019. http://hdl.handle.net/11384/85883.

Testo completo

Gli stili APA, Harvard, Vancouver, ISO e altri

Abstract (sommario):

One prominent feature of brain computation is the excitation inhibition balance (E/I balance) that represents one of the main homeostatic functions of the brain. Its aim is to maintain the neural circuits in a narrow and safe range of action. Within this range, the brain network can receive and analyze sensory inputs and produce a modulated output, proportional to the stimuli intensity. Any imbalance in this equilibrium leads to abnormal responses to external stimuli and results in pathological behavior. Indeed, neurological pathologies known for featuring a deep alteration of the E‐I balance are epilepsy and autism, which often occur together in the same patient. Several human genetic syndromes caused by alterations of genes involved in neural development feature signs like autism and epilepsy. Thus, they represent important cases for studying and understanding the role of these single altered genes in the development and regulation of the brain balance. In return, we hope that this knowledge of these genes and more generally of human brain network can be useful in treating the patients affected by these conditions and can help us improve their quality of life. In my work, I studied the regulation of the E/I balance in mouse models of neurological diseases from three different points of view. In the first set of experiments, I studied the E/I balance in a focal model of epileptiform activity. This model is produced by the local application of bicuculline to the mouse cortex. Bicuculline is a competitive GABAA receptor antagonist that, when applied, leads to the development of persistent and periodic interictal spikes at the injection site, while activity appears to be normal in nearby areas that are not reached by bicuculline. In our experiments, we showed that, even in the apparently normal area, there is a disruption of cortical computation. Specifically, the disruption occurs whenever an interictal spike is generated in the epileptic focus. This can have important impact on our understanding of epilepsy and of its treatment since interictal spikes are a common feature not only of epileptic patients, but can also appear in non‐epileptic subject, apparently without any consequence. From our results, we concluded that interictal activity can actually interfere with brain operation not only in the center of the epileptiform activity, but also in the connected areas, where the E/I balance is not directly disrupted. These results provide an example of the fact that apparently non‐symptomatic interictal spikes can affect brain computation. The second experimental model that I studied is a mouse model for a specific human genetic disease: the Phelan‐McDermid syndrome. This is a developmental disease, caused by a genomic deletion at site 22q13. The main suspect for causing the disease is one gene, Shank3, which encodes for a scaffold protein localized in the post‐synaptic density of glutamatergic synapses. In this model, we studied the computation of visual stimuli and we found an alteration of the contrast‐response curve. This is a defining relationship of visual processing: it is the transfer function that converts the visual input into a neural output. This means that to each intensity of visual stimulation corresponds a certain intensity of the neural response, of the visual cortex. We determined that, in Shank3 mutant mice, this curve was altered and showed an increased response to less intense stimuli and showed also a poor modulation of responses to high‐contrast stimulations. An interpretation of this can be that these mice are more sensitive to low‐contrast stimuli, but completely lose the ability of telling apart different high‐contrast stimuli from each other. Therefore, the Phelan McDermid mouse becomes “blinded” by weak stimulations as if they were seeing strong stimulus. Finally, we studied the behavior of the chloride ion in a drug‐induced epileptic seizure model. Chloride ion is of pivotal importance in neurons were the activation of ionotropic GABA and glycine receptors, which increase chloride membrane conductance in response to GABA or glycine release respectively. The intracellular concentration of chloride ions decides what is the effect of GABA release. Traditionally, ionotropic GABA receptors activation was thought to be inhibitory only, but the excitatory or inhibitory nature of these receptors is determined by the intracellular concentration of chloride ions. This concentration in normal adult neurons is thought to be around 5 mM: at this concentration, the effect of the activation of GABA receptors is an inhibition of the postsynaptic element. We investigated if the chloride concentration can be varied under extreme pathologic conditions as during epileptic seizures in a drug induced mouse model. In these animals, the epileptic seizures were produced by local administration of 4‐aminopyridine (4‐AP), a potassium channel antagonist. The effect of 4‐AP is to cause accumulation of chloride ions in neurons and this suggests that, in epileptic crisis, the role of inhibitory neurons can actually favor excitation.

15

Chu, Chia-ying, e 朱家瑩. "Expression of Heterologous Promoter Sequencesin Fish Cell line and Zebrafish Embryos". Thesis, 1997. http://ndltd.ncl.edu.tw/handle/10724015166858123007.

Testo completo

Gli stili APA, Harvard, Vancouver, ISO e altri

Abstract (sommario):

碩士
國立臺灣大學
動物學系
85
In order to study the gene regulation in cell lines and embryos, several expression vectors containing different heterologous promoters as well as lacZ reporter gene were constructed. The promoter regions were obtained from a variety of fish genes including common carp (Cyprinus carpio) JAK1 and cdc2 genes as well as the round-spotted pufferfish (Tetraodon fluviatilis ) JAK1, RET and c-fos genes. The results of promoter assay were different in cell lines and in embryos. When transfected into carp CF cell line, pf-fos promoter displayed strong lacZ activity, about 15% of that of pCMVβ, While pf-JAK1 promoter had only 7% of that of pCMVβ. When these constructs were injected into zebrafish (Danio rerio) embryos at one-cell- stage, the pf- JAK1 promoter displayed stronger expression activity than that of pf-fos promoter. Analyzing the expression pattern of 24 -h (hours after fertilization at 28℃) embryos, the β-gal- expressed cells of pf-JAK1 and c-JAK1 promoters were predominantly located in notochord and muscle in the trunk segments of embryos, whereas those of c-cdc2 promoter were located ubiquitously. The majority of expressing cells of pf-RET promoter was located in the head and skin region. We also performed deletion analysis of c-cdc2 promoter and a positive regulation region between -211 and -146 was defined. A sequence in the region (5'-AAATT AACAAA-3') has high similarity with rat cdc2 enhancer element (-276AA GTTACAAA-267). With the goal of detecting gene expression in the embryo at different developmental stages, the green fluorescent protein (GFP) from the jellyfish (Aequorea victoria) was chosen as reporter gene. GFP fluorescence can be observed non- invasively in living cells without any substrate or cofactor. A GFP expression vector driven by c-JAK1 promoter was constructed and microinjected into zebrafish embryos. At 44-h and 72-h after injected, the green fluorescence was visible in muscle and notochord. Our data indicated that the transient expression system, by means of transgenic zebrafish, could be used as rapid analysis of promoter activity and gene regulation in vivo. Utilizing this system, we found that some promoters were expressed ubiquitously whereas other promoters were expressed at restricted area. Thus, analysis of promoter activity would be useful for further studies.

16

Chan, Ju-Chien, e 詹茹潔. "The persistent infection of fish nodavirus in grouper brain cell line". Thesis, 2011. http://ndltd.ncl.edu.tw/handle/82159564855597487681.

Testo completo

Gli stili APA, Harvard, Vancouver, ISO e altri

Abstract (sommario):

碩士
國立臺灣大學
動物學研究所
99
Grouper survivors after nervous necrosis virus (NNV) acute infection will become persistently infected. In order to reveal the relationship of grouper Mx protein and the persistent infection of NNV, a cell line GB was established from the brain tissue of grouper survivors. NNV was found in GB cells by RT-PCR, and NNV protein was only detectable in the cytoplasm of a few GB cells by immunohistochemistry staining, suggesting that only a few cells were acutely infected and most cells were likely protected. After serial treatments of NNV-specific polyclonal antibodies, GB cells became a NNV-free cell line which was named as cured GB (cGB). The expression of grouper Mx (GMx), a downstream protein of interferon response, was detected in GB cells, NNV-infected cGB cells and poly I:C-transfected cells, but was abscent in cGB cells, indicating that IFN response existed in GB cells, and IFN response and Mx expression was inducible in cGB cells by NNV infection and poly I:C transfection. When cGB cells were infected by NNV with low MOI, all characteristics of persistent infection in GB cells reappeared in infected cGB cells. In addition, NNV titer proliferated in poly I:C-transfected cGB cells were lower than that in cGB cells, indicating that the mechansim of NNV persistence in GB cells possibly mediated through IFN and Mx protein. By the way, NNV. At 24 h post-infection, only viral RNA-dependent RNA polymerase (RdRp) was found to be colocalized with Mx at perinuclear area, suggesting that Mx might interfere with NNV replication by interaction with RdRp.

17

Borges, Alice Da Cruz Madeira. "Molecular cytogenetic characterization of murine cell line SEWA". Master's thesis, 2017. http://hdl.handle.net/10451/35903.

Testo completo

Gli stili APA, Harvard, Vancouver, ISO e altri

Abstract (sommario):

Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2017
A hereditariedade e as alterações estruturais dos cromossomas são dois temas ligados à Citogenética Clínica que apresentam maior impacto aquando da tomada de decisão relativa a diagnósticos tumorais e pré/pós natais. Através de métodos de hibridação de fluorescência in situ, desenvolvidos ao longo dos anos, é possível obter informações relativas a alterações estruturais e variações numéricas no cariótipo celular, permitindo retirar conclusões relativamente à origem e prognóstico de cancros, e à viabilidade de fetos quando uma determinada doença congénita está presente numa família. Para que estes estudos sejam passiveis de ser realizados são necessárias linhas celulares modificadas. A linha celular SEWA é uma das mais usadas a nível de estudos carcinogénicos. Desenvolvida inicialmente a partir de um sarcoma osteogénico induzido por um polioma-vírus, o seu material carcinogénico foi posteriormente transformado em tumor ascítico e transplantado para ratinhos da linhagem A.SW. A sua importância para os estudos sobre tumores prende-se no facto de esta linha celular ser caracterizada pela presença in vivo de cromossomas double minute, de cromossomas C-bandless e homogeneously staining regions. Estas estruturas cromossómicas contêm o oncogene c-myc, cujo grau de amplificação está diretamente correlacionado com a gravidade do tumor. No entanto, até à data, nenhuma caracterização do seu cariótipo foi publicada. Este trabalho visa apresentar um possível cariótipo para esta linha celular, com uma descrição detalhada das suas alterações estruturais e numéricas. Para tal, técnicas como MCB, SKY e aCGH foram usadas. Qualquer uma destas técnicas é derivada de uma tecnologia desenvolvida no final dos anos 60, denominada de hibridização de fluorescência in situ (FISH). Neste tipo de métodos, diferentes sondas são marcadas com diferentes marcadores fluorescentes de modo a que seja possível visualizar regiões específicas do genoma. Os resultados, obtidos através de processamento de imagem, seriam posteriormente usados para encontrar homólogos no genoma humano e associar essas alterações a um tumor humano. A presença de cromotripsis, estruturas comuns em tumores agressivos, que ocorrem nas etapas iniciais de carcinogénese, seria também indício de mau prognóstico na sobrevivência ao cancro. Estudos como este permitem que modelos de origem tumoral sejam desenvolvidos, possibilitando um prognóstico mais prematuro relativamente ao desenvolvimento e cura de determinados tumores. Neste trabalho, 30 metafases foram analisadas, tanto para o método SKY, como para o MCB, sendo que no último a análise foi realizada cromossoma a cromossoma. No entanto, devido à má qualidade dos resultados obtidos e a alguma inexperiência do operador, não foi possível retirar conclusões concretas relativamente às alterações cromossómicas encontradas. Apenas se concluiu que as células SEWA apresentam um cariótipo instável, com diferentes alterações entre metafases. Além disso, resultados entre métodos não coincidiram o que levou à impossibilidade de criar um cariótipo representativo da população celular analisada, nem à descrição de breakpoints, que teriam significado para determinar uma futura homologia com o genoma humano. Será então necessária uma reavaliação dos resultados para que novas conclusões sejam obtidas.
Inheritance and structural changes of chromosomes are two themes related to Clinical Cytogenetics that present greater impact when making decisions regarding tumor and pre and post-natal diagnoses. Through in situ hybridization methods developed over the years, it is possible to obtain information on structural changes and numerical variations in the cellular karyotype, allowing investigators to draw conclusions regarding the origin and prognosis of cancers, and the viability of fetuses when certain congenital diseases are present in a family. The SEWA cell line, initially developed from osteogenic sarcoma induced by a polyoma virus, is one of the most widely used in carcinogenic studies. However, to date, no characterization of its karyotype has been published. This work aims to present a possible karyotype of this cell line, with a detailed description of its structural and numerical changes. For such, techniques like MCB, SKY and aCGH were used. The results would then be used to find homologs in the human genome and associate those changes with a human tumor. Studies like this allow models of tumor genesis to be developed, allowing an earlier prognosis regarding the development and cure of certain tumors. In this work, 30 metaphases were analyzed, both for SKY method and MCB. In the last one, the analysis was performed on each of the chromosomes. However, due to the poor quality of the results obtained and some inexperience of the operator, it was not possible to draw concrete conclusions regarding the chromosomal alterations that were found. It was only concluded that SEWA cells present an unstable karyotype, with several changes present in only one metaphase. Moreover, results between methods did not coincide, which led to the impossibility of creating a representative karyotype of the cell population and describing the breakpoints that would have meaning in homology with the human genome. It will then be necessary to re-evaluate the results so that new conclusions can be obtained.

18

紀璟叡. "Stable Expression Of Birnavirus Antisense RNA In Fish Cell Line CHSE-214". Thesis, 1995. http://ndltd.ncl.edu.tw/handle/39097116122496976705.

Testo completo

Gli stili APA, Harvard, Vancouver, ISO e altri

Abstract (sommario):

碩士
國立海洋大學
水產食品科學研究所
83
Infectious pancreatic necrosis virus (IPNV) is an unportant viral pathogen to cause infectious fish disease. In Taiwan, IPNV has caused great damages and loss in the aquaculture of tilapia, ell, milk fish and clam. Therefore, how to effectively control the IPNV infection is an important issue to the aquacultures, Since using the antisense method to inhibit the virus replication has advantages of high specificity and low biotoxicity, in this study we constructed several antisense expression vectors, CMV21 (A), SV21 (A) and RSV21 (A), with the cytomegalovirus (CMV) immediately promoter or the SV40 early promoter as a promoter and the IPNV - EOS cDNA fragment, PB21, complementary to the B fragment gene, VPI, as an insert. These vectors and other vectors constructed before by our laboratory colleagues were introduced into the CHSE - 214 cells and 70 stable transfecied cell lines were selected. The result of virus titration determined on 27 cell lines showed that the transfected cell line SA21 (A) -3 has the best virus inhibition effect, whose virus titer decreased 2 log values. According to the result of the Southern blotting hybridization, the transfected DNA can not be detected in only three lines. As we extracted cellular proteins before virus infection and at 2, 4, 6, 8, 12 and 24 hours post - infection (at M. O. I. 1), and used the IPNV - EIS polyclonal antibody for the Western blotting hybridization, we found that the peak of virus protein 1 (VPI) production showed at 6 hour post - infection in the control, while in the cell line SV21 (A) -3, the peak was found at 8 hours post - infection and its highest yield was 33.17% less than the control. These results suggest that the antisense RNA complementary to VPI, of colony SV21 (A) can slow down and reduce the VPI production.

19

Han, Wan-Jyuan, e 韓宛娟. "The Studies of Anti Grouper Virus Infection by Type I Interferon in Fish Cell Line". Thesis, 2013. http://ndltd.ncl.edu.tw/handle/20062643186371966461.

Testo completo

Gli stili APA, Harvard, Vancouver, ISO e altri

Abstract (sommario):

碩士
國立臺灣海洋大學
水產養殖學系
101
The causative of viral nervous necrosis are nervous necrosis virus (Betanodavirus) and there are over 30 species of cultured marine fish could be infected. In Taiwan nervous necrosis virus is the most devastating disease which particularly occurred in grouper’s larvae, where the mortality rate is more than 90%. Conversely, iridovirus has been confirmed that could cause systemic infection in over more than 20 species of fishes. It has high infectivity and high mortality on fishes. Both of these two viruses lead to economic lost in aquaculture industry in Taiwan. Hence, developing an efficient antiviral strategy has become an important issue. Type I interferon system is a rapid and powerful antiviral defense mechanism in vertebrates. When the cell infected by virus interferon well induction of anti-viral molecules, such as Mx protein, and several apoptotic pathways. In this study, we produced grouper type I interferon in Escherichia coli (E. coli) system. Investigating that the type I interfere antiviral function affected in nervous necrosis virus and iridescent virus, as well as discussing cell protective effect treated IFN in different time and different mode. We found that Mx gene expressed after 12 hr when synthetic type I interferon treated cells, further the cytopathic effect (CPE) was delayed increased for survival of cells. Viruses’ replication was also inhibited as shown by real-time RT-PCR assay. In nervous necrosis virus infection, IFN pre- treated can reduce the virus titer in the range of 10 to 30 fold whereas in grouper iridovirus virus infection, IFN pre- treated can reduce the virus titer in the range of 5 to 50 fold.

20

Chun-ju, Tai, e 戴君如. "The Inhibitions of IPNV Induced Apoptosis by Its VP3 Antisense RNA Expression in Fish Cell Line". Thesis, 2000. http://ndltd.ncl.edu.tw/handle/15429056548608768136.

Testo completo

Gli stili APA, Harvard, Vancouver, ISO e altri

Abstract (sommario):

碩士
國立臺灣大學
漁業科學研究所
88
Abstract IPNV (Infectious Pancreatic Necrosis Virus) is a double-stranded RNA viruse which has been shown to be a major cause of many contagious and widespread fish diseases. IPNV belongs to Birnaviridae virus family and is also a pathogen of many economically important fishes in Taiwan. The IPNV-E1S (Eel No.1 Spleen) virus strain was isolated from the spleen of diseased Japanese eel (Anguilla japonica) and is an Ab serotype of Aquatic Birnavirus. Recent study has established apoptosis as a major cause for the observed IPNV-induced cytopathic effects in infected fish cells. IPNV-infected CHSE-214 cells displayed all previously known morphological and biochemical hallmarks of apoptosis. At present, the apoptosis-inducing activity of IPNV has not been definitively assigned to any IPNV factors. Nevertheless, there was evidence implicating VP3 as such a factor. To identify the unknown apoptosis-inducing element of IPNV, CHSE-214 cells stably expressing various antisense RNAs (A2, A4, and A7, complement to nt 501-711, 285-711, and 1-711, respectively) against VP3 were established. All antisense RNA expressing cells showed increased resistance to IPNV-induced apoptosis in viral challenge experiments (MOI 1). Further analyses showed that VP3 protein expression was suppressed, Mcl-1 expression was longer sustained, and DNA fragmentation was reduced in these antisense RNA expressing cells during early viral replication cycle. These results suggest that the antisense RNAs could knock down the VP3 expression and VP3 is an apoptosis-inducing factor responsible for the IPNV-induced apoptotic cell death.

21

Wu, Yu-Chi, e 吳育騏. "Persistent Infection of Fish Nodavirus in Barramundi Brain Cell Line and the Antiviral Mechanism of Barramundi Mx Protein". Thesis, 2008. http://ndltd.ncl.edu.tw/handle/72383820037327001933.

Testo completo

Gli stili APA, Harvard, Vancouver, ISO e altri

Abstract (sommario):

博士
國立臺灣大學
動物學研究所
96
The BB cell line derived from the brain tissue of a barramundi (Lates calcarifer) that survived nervous necrosis virus (NNV) infection is persistently infected with NNV. To elucidate whether interferon (IFN) plays a role in the mechanism of NNV-persistent infection in BB cell line, a virus-negative control cell line was obtained by treating BB cells with NNV-specific rabbit antiserum for 5 subcultures. After the treatment, NNV titer or RNA or capsid protein was no longer detected in the cured BB (cBB) cells. Expression of Mx gene, encoding a type I IFN-inducible antiviral protein, was found in BB cells and cBB cells following NNV infection, but not in NNV-free cBB cells. Moreover, expression of Mx gene and antiviral activity against NNV were induced in cBB cells by the treatment with MAb-neutralized BB cell supernatant. Furthermore, NNV persistent infection was induced again in cBB cell culture if multiplicity of infection (MOI) was low (≦1). These experimental results indicated that IFN-like cytokines existed in the culture supernatant of BB cells, and IFN-induced response played an important role in protecting the majority of cells from virus lytic infection and regulating NNV persistence in the BB cell line. We obtained a full-length cDNA clone for the Mx gene of barramundi (Lates calcarifer), using RACE (rapid amplification of cDNA ends) polymerase chain reaction (PCR) amplification of RNA extracted from a barramundi brain cell line cBB. The Mx cDNA of 2.2 kb contains an open reading frame (ORF) of 1875 nucleotides encoding a protein of 624 amino acids. The predicted barramundi Mx protein is 71.4 kDa and contains a tripartite guanosinetriphosphate (GTP)-binding motif at the amino terminal and a leucine zipper at the carboxyl terminal, characteristic of all known Mx proteins. Poly I:C-transfection induced the expression of Mx gene in cBB cells, and the induction level at 28 ℃ was higher than that at 20 ℃. Moreover, Mx gene expression was also induced by viral infection, including fish nodavirus, birnavirus, and iridovirus. Among these, nodavirus was a stronger inducer than the other two viruses. Using an antiviral activity assay, we revealed that poly I:C-transfected cBB cells had antiviral activity against fish nodavirus and birnavirus, but not iridovirus. Furthermore, the replication of nodavirus and birnavirus could be restored after the expression of Mx gene was down-regulated by siRNA. Therefore, these results indicated that the expression of barramundi Mx gene was able to inhibit the proliferation of fish nodavirus and birnavirus. The expression levels of NNV RNA-dependent RNA polymerase (RdRp) and capsid protein were found to increase in NNV-infected cBB cells within 24 h post-infection (hpi), but later decrease as soon as the expression of barramundi Mx protein. Moreover, NNV capsid protein, RNA2, and viral titer all elevated in the cBB cells when barramundi Mx protein expression was down-regulated by siRNA. Barramundi Mx protein was observed to colocalize with NNV RdRp at the perinuclear area in NNV-infected cBB cells by immunofluorescence staining, and was co-precipitated with NNV RdRp by immunoprecipitation assay. Furthermore, the complexes of barramundi Mx protein and NNV RdRp could colocalize with lysosomes, and then NNV RdRp would be degragded by the lysosome system. Therefore, it was suggested that barramundi Mx protein suppressed NNV RNA synthesis by sequestration of NNV RdRp to the perinuclear area for the following degradation in the lysosomes.

22

Wu, Yu-Chi. "Persistent Infection of Fish Nodavirus in Barramundi Brain Cell Line and the Antiviral Mechanism of Barramundi Mx Protein". 2008. http://www.cetd.com.tw/ec/thesisdetail.aspx?etdun=U0001-2907200803431800.

Testo completo

Gli stili APA, Harvard, Vancouver, ISO e altri

23

Lin, Chia-Hua, e 林佳樺. "Establishment and characterisation of a fibroblast cell line derived from the heart of balloon fish, Diodon holocanthus (Linnaeus)". Thesis, 2013. http://ndltd.ncl.edu.tw/handle/35662337208671421886.

Testo completo

Gli stili APA, Harvard, Vancouver, ISO e altri

Abstract (sommario):

碩士
國立澎湖科技大學
水產資源與養殖研究所
101
The establishment and partial characterisation of a continuous cell line from the heart of balloon fish Diodon holocanthus (Linnaeus) are described. The cell line, designated BH-1, has been subcultured for more than 85 passages since its initiation in July of 2010. Analysis of mitochondrial cytochrome b gene sequences revealed 99% match for BH-1 to known D. holocanthus mitochondrial DNA sequences (GenBank accession no. AP009177). The BH-1 cell line was susceptible to hard clam reovirus (HCRV). It was optimally maintained at 28 °C in Leibovitz L-15 medium with 20% fetal bovine serum (FBS). Propagation of BH-1 cells was serum dependent; it exhibited poor growth between 2.5 and 10% FBS, relatively good growth at 15%, and maximum growth occurred with 20%. Fish serum is easy to be acquired in Penghu Island and has found to be able to prompt cell growth. Growth medium supplemented with extra 0.5 or 1% cobia serum (CS) for 6 days, the propagation of BH-1 cells was better than control group which was cultured in free of cobia serum. The results showed that the growth medium supplemented with 1% or 0.5% cobia serum promoted cell growth significantly.

24

Kawano, Atsushi. "Developing and characterizing a salmonid intestinal epithelial cell line for use in studies of inflammation in the fish gastrointestinal tract". Thesis, 2009. http://hdl.handle.net/10012/4390.

Testo completo

Gli stili APA, Harvard, Vancouver, ISO e altri

Abstract (sommario):

An intestinal cell line from rainbow trout, Oncorhynchus mykiss, was developed and challenged against several bioactive components. Primary cultures initiated from the distal segment produced the cell line, RTgutGC. RTgutGC showed optimal growth in L15 supplemented with 10-20% fetal bovine serum (FBS) at room temperature. RTgutGC has undergone over 100 passages and stained minimally for β-galactosidase, suggesting this to be an immortal cell line. Late passage cultures gave a consistent polygonal morphology with distinct borders. RTgutGC stained positive for alkaline phosphatase (AP) under certain culture conditions, hence may produce intestinal-specific alkaline phosphatase (IAP). Lipopolysaccharide (LPS) was used as a model microbial endotoxin for determining the sensitivity of the cells to a natural ligand in the gastrointestinal tract (GIT). Exposure of LPS was compared between RTgutGC and two mammalian intestinal cell lines (HT-29 and Caco-2). LPS induced cell death in RTgutGC, potentially through an alternative pathway seen in higher vertebrate response. Cytotoxicity of LPS against RTgutGC, seeded at normal density, was reduced in the presence of glutamine compared to L15 alone (t test, p≤ 0.05). RTgutGC seeded at a super density, where AP was strongly expressed, also showed less toxicity towards LPS. Two isoforms of tumor necrosis factor alpha (TNF-α) transcripts were up-regulated after LPS treatment in RTgutGC. Six rainbow trout cell lines, including RTgutGC, showed constitutive transcript expression of several immune-related genes: Major Histocompatibility (MH) class II α and ß. When MH activity was examined at the protein level, the cell lines showed constitutive expression of MH class I proteins, but not for MH class II molecules. RTS11, a rainbow trout spleen monocyte/ macrophage-like cell line, was the only line to express all MH transcripts and proteins. The utility of the anti-rainbow trout MH protein sera was demonstrated by exposing RTgutGC to poly IC. After a 3 day treatment, RTgutGC showed up-regulation of β2m protein expression. Thus, the cellular and immunological responses in fish intestinal cells can be modeled using the methods presented in this study.

25

Yeh, Shang-wei, e 葉上暐. "Neural marker expression of a clonal cell line (ARB8-2) isolated from the brain of cichlid fish (Aequidens rivulatus)". Thesis, 2014. http://ndltd.ncl.edu.tw/handle/18377282290998947276.

Testo completo

Gli stili APA, Harvard, Vancouver, ISO e altri

Abstract (sommario):

碩士
國立高雄大學
生命科學系碩士班
102
A clonal cell line (ARB8-2) isolated from the brain of cichlid fish (Aequidens rivulatus) was investigated by reverse transcription polymerase chain reaction (RT-PCR),Western blot, immunocytochemistry, PCR and Real-time PCR were utilized to characterize the molecular expression of ARB8-2 cell line. The results showed that the ARB8-2 express radial glia markers such as nestin and brain lipid binding protein (BLBP), astrocyte markers such as glial fibrillary acidic protein (GFAP), glutamine synthetase (GS) and vimentin, oligodendrocyte progenitor cell marker such as A2B5, neuron markers such as tyrosine hydroxylase (TH), epithelial cell markers such as gap junction connexin 43 (Cx43) and tight junction occludin, melanocyte markers such as tyrosinase, tyrosinase-related protein-2 (TRP-2), microphthalmia-associated transcription factor (MITF), SLC45A2 and keratinocyte-associated transmembrane protein 2 (KCT-2) and retinal epithelial marker retinal pigment epithelium-specific 65 (RPE65). However, the ARB8-2 also express homeodomain-containing transcription factors NKX 2.3 and NKX 6.2 and GATA 3. The results indicated that ARB8-2 might be the neural stem cell.

26

Tarasco, Marco. "Screening natural resources for mineralogenic and osteogenic bioactivities using in vitro and in vivo fish systems". Master's thesis, 2016. http://hdl.handle.net/10400.1/8653.

Testo completo

Gli stili APA, Harvard, Vancouver, ISO e altri

Abstract (sommario):

Dissertação de Mestrado, Biologia Marinha, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2016
Bone disorders affect millions of people worldwide, and available treatments have only a limited efficacy and/or bring undesirable side effects. There is therefore the need for novel compounds with bone anabolic properties. Due to its technical advantages, fish have been successfully used in biomedical and pharmaceutical research to screen for osteogenic compounds. The aim of this work was to evaluate mineralogenic/osteogenic performance of semi-purified fractions and purified molecules from terrestrial plants (cardoon and eucalyptus) and cyanobacteria. VSa13 cell line established from gilthead seabream (Sparus aurata) vertebra and capable of in vitro mineralization was used to assess the mineralogenic potential of the extracts, while zebrafish operculum was used to develop and establish a reliable in vivo model to screen molecules for osteogenic activity. Extracts from cardoon, eucalyptus and some cyanobacteria exhibited some moderate mineralogenic effect while triterpenic acids purified from eucalyptus strongly increased mineral deposition. Pro-mineralogenic compounds were further analysed for their bone anabolic action on zebrafish operculum and lipophilic cardoon extract, ursolic acid, oleanolic acid and ethyl acetate fraction of cyanobacteria strain #13 were found to promote an osteogenic effect. While the osteogenicity of ursolic acid has already been reported in mouse, the action of oleanolic acid and cyanobacteria extract on bone formation is revealed here for the first time. The use of a double transgenic line expressing fluorescent proteins under the control of osterix/sp7 and osteocalcin/oc2 promoters to get insights into mechanisms underlying the osteogenic activity of natural compounds has been explored. In vitro and in vivo data generated within the scope of this work have not only demonstrated the potential of natural resources to provide molecules with mineralogenic/osteogenic activity that may be used in pharmaceuticals or nutraceuticals but also the suitability of fish systems to screen for these molecules.
O tecido ósseo é um tipo especializado de tecido conjuntivo constituído por células e por uma matriz extracelular, que possui como característica única a capacidade de promover a sua mineralização,, o que confere a este tecido uma extrema dureza, permitindo-lhe desempenhar importantes funções quer de suporte mecânico aos músculos, possibilitando o movimento, quer de proteção de órgãos internos, constituindo também uma reserva de iões de cálcio e de fosfato. As patologias mais importantes que afectam o esqueleto incluem doenças metabólicas, que afectam o crescimento através de alterações na formação e remoção óssea, fracturas, deformações várias,, infecções bacterianas e tumores. Entre as doenças metabólicas que afectam milhões de pessoas em todo o mundo e que resultam do desequilíbrio entre a atividade osteoblástica (formação óssea) e a atividade osteoclástica (reabsorção óssea) estão a osteoporose, a osteopetrose, o raquitismo e a doença óssea de Paget. No entanto os tratamentos usados são ineficazes e/ou estão associados a efeitos secundários indesejáveis existindo a necessidade de descobrir novos compostos com a capacidade de reverter os sintomas dessas patologias no osso. Os recursos naturais representam uma fonte valiosa de moléculas bioativas, em particular de compostos osteogénicos. A análise em grande escala de extratos ou moléculas com potenciais efeitos osteogénicos requer o uso de ferramentas in vitro e in vivo optimizadas Organismos aquáticos como o peixe-zebra (zebrafish) têm sido utilizados com sucesso nas investigações biomédica e farmacêutica devido às suas vantagens técnicas em relação a modelos animais mais clássicos, como o ratinho. Por exemplo o peixe-zebra possui um ciclo de vida mais curto, gera um elevado número de descendentes, tem o genoma sequenciado, os embriões são transparentes e desenvolvem-se externamente. Além disso, os peixe-zebra adultos também podem ser facilmente visualizados e manipulados experimentalmente e existem mutantes/transgénicos que permitem a análise da expressão de multiplos genes através de microscopia de fluorescência. O objectivo principal deste trabalho é avaliar a capacidade mineralogénica/osteogénica de fracções semi-purificadas e de moléculas purificadas de plantas terrestres, como o cardo ou o eucalipto, e de cianobactérias. A linha celular VSa13 obtida a partir de vértebras de dourada (Sparus aurata), que possui a capacidade de mineralização in vitro, foi usada para analisar o potencial mineralógenico dos extratos usados, e o opérculo do peixe-zebra foi usado para desenvolver e estabelecer um modelo in vivo adequado para analisar os efeitos de moléculas com atividade osteogénica (através do uso do calcitriol). Os nossos resultados demostraram a existência de um efeito mineralogénico moderado usando concentrações não tóxicas de extratos de cardo, de eucalipto e de algumas cianobactérias, enquanto que ácidos triterpénicos purificados a partir de amostras de eucalipto (como por exemplo o ácido betulónico, ursólico e oleanólico) aumentam até 6 vezes a deposição de mineral na matrix extracelular da linha celular VSa13. Os nossos resultados mostraram ainda que in vitro, a cinaropicrina, uma lactona sesquiterpénica abundante nos extratos de cardo, promovia um efeito anti-mineralogénico, o que foi mais tarde comprovado utilizando o nosso modelo in vivo. O efeito de compostos pro-mineralogénicos foi posteriormente analisado através do estudo do opérculo do peixe-zebra e os nossos resultados demonstraram que os extratos lipofílicos do cardo, como o ácido ursólico, o ácido oleanólico e a fracção de acetato de etílo da espécie #13 de cianobactérias, possuem efeitos osteogénicos. Apesar da capacidade osteogénica do ácido ursólico já ter sido analisada e comprovada por outros grupos usando um modelo animal de ratinho, a ação do ácido oleanólico, da cinaropicrina e de extratos de cianobactérias na formação óssea nunca tinha sido descrita. O uso de uma linha de duplos transgénicos que expressam uma proteína fluorescente sob o controlo dos promotores do osterix/sp7 ou da osteocalcina/oc2 tem sido determinante para tentar perceber os mecanismos responsáveis por esta atividade osteogénica e sendo a mesma abordagem atualmente aplicada ao estudo do efeito dos ácidos triterpenicos. Os dados gerados a partir das ferramentas in vitro e in vivo utilizadas neste trabalho não só demonstraram o potencial de recursos naturais (terrestres ou marinhos) para a descoberta de moléculas com efeito mineralogénico/osteogénico potencialmente relevantes para as indústrias farmacêutica ou alimentar, como também evidenciaram a importância do uso de animais modelos como o peixe-zebra para a análise do efeito destas moléculas.

27

George, Githa Ann. "Establishment and characterisation of cell lines from the caerulean damsel, Pomacentrus caeruleus". Thesis, 2017. http://eprints.cmfri.org.in/13915/1/Githa%20Ann%20George_2017_Thesis_Pomacentrus%20caeruleus.pdf.

Testo completo

Gli stili APA, Harvard, Vancouver, ISO e altri

Abstract (sommario):

Cell lines and primary cell cultures from fish tissues have been used for research in disease diagnosis and cytotoxicity evaluation of environmental pollutants. The principal aim of this work was the development of continuous cell lines from the caerulean damsel, Pomacentus caeruleus. To achieve this, tissues were first sourced from donor fishes, devoid of contaminants using standardised disinfection protocols. Primary cultures were then initiated and suitable media, additives, dissociation reagents as well as optimum incubation temperature were determined. Successful primary cultures were subcultured and passaged to derive continuous cell lines which were cryopreserved for long term storage. The continuous cell lines established were characterised by immunotyping using cell type markers and authenticated to confirm the species of origin using previously established barcoding techniques. Preliminary applications such as gene transfection studies and cytotoxicity assays using bacterial extracellular products were done in addition to ensuring that the cultures and cryostocks were contamination-free.

28

Pham, Phuc Hoang. "Using cell lines to study factors affecting transmission of fish viruses". Thesis, 2014. http://hdl.handle.net/10012/8112.

Testo completo

Gli stili APA, Harvard, Vancouver, ISO e altri

Abstract (sommario):

Factors that can influence the transmission of aquatic viruses in fish production facilities and natural environment are the immune defense of host species, the ability of viruses to infect host cells, and the environmental persistence of viruses. In this thesis, fish cell lines were used to study different aspects of these factors. Five viruses were used in this study: viral hemorrhagic septicemia virus (VHSV) from the Rhabdoviridae family; chum salmon reovirus (CSV) from the Reoviridae family; infectious pancreatic necrosis virus (IPNV) from the Birnaviridae family; and grouper iridovirus (GIV) and frog virus-3 (FV3) from the Iridoviridae family. The first factor affecting the transmission of fish viruses examined in this thesis is the immune defense of host species. In this work, infections of marine VHSV-IVa and freshwater VHSV-IVb were studied in two rainbow trout cell lines, RTgill-W1 from the gill epithelium, and RTS11 from spleen macrophages. RTgill-W1 produced infectious progeny of both VHSV-IVa and -IVb. However, VHSV-IVa was more infectious than IVb toward RTgill-W1: IVa caused cytopathic effects (CPE) at a lower viral titre, elicited CPE earlier, and yielded higher titres. By contrast, no CPE and no increase in viral titre were observed in RTS11 cultures infected with either genotype. Yet in RTS11 all six VHSV genes were expressed and antiviral genes, Mx2 and Mx3, were up regulated by VHSV-IVb and -IVa. However, replication appeared to terminate at the translational stage as viral N protein, presumably the most abundant of the VHSV proteins, was not detected in either infected RTS11 cultures. In RTgill-W1, Mx2 and Mx3 were up regulated to similar levels by both viral genotypes, while VHSV-IVa induced higher levels of IFN1, IFN2 and LGP2A than VHSV-IVb. The second part of the thesis examined the ability of two Ranaviruses, GIV and FV3, to infect non-host fish cells. This is referred to as cellular tropism and is one of many host-virus interaction events required to established successful infection in new organisms. Grouper iridovirus (GIV), belonging to the Ranavirus genus of the Iridoviridae family, was demonstrated to differentially express viral genes and induce apoptosis in three non-host fish cell lines rainbow trout monocyte/macrophage (RTS11), Chinook salmon embryon (CHSE-214) and fathead minnow Epithelioma papulosum cyprinii (EPC). These cells were challenged with GIV and virus entry into all three cell lines was confirmed by the expression of viral immediate early genes. The expression of the late major capsid protein gene was detected in CHSE-214 and EPC, but not in RTS11, suggesting an earlier termination in the viral replication cycle in RTS11. Approximately 12 h after infection with GIV, cell death was prominent in all three non-host cell lines. Death was later confirmed to be apoptosis by the presence of chromosomal DNA fragmentation and phosphatidylserine externalization. To determine whether apoptosis was protein related or gene expression related, the three cell lines were infected with heat-inactivated GIV and UV-treated GIV (GIVUV). The heat inactivation abolished apoptosis in all three cell lines, but each cell line responded differently to GIVUV. Relative to GIV, GIVUV caused no apoptosis in CHSE-214, decreased apoptosis in RTS11, and increased apoptosis in EPC. These results suggest that early GIV gene expression was needed for apoptosis in CHSE-214 but impeded apoptosis in EPC. At the cellular level, only EPC was a permissive host as EPC was the only cell line of the three capable of producing a moderate increase in virus titre. The three non-host cell lines present a good system for potentially identifying different components of GIV-induced apoptotic pathways in future studies. Rainbow trout are not highly susceptible to frog virus 3 (FV3) induced diseases, and had been suggested to be a potential carrier for the virus. To determine which rainbow trout cell types are permissive for FV3 and act as a potential source for virus replication in vivo, the ability of rainbow trout cell lines from gonads (RTG-2), skin (RTHDF), liver (RTL-W1), gills (RTgill-W1), intestine (RTgut-GC) and spleen (RTS11), and primary leukocyte cultures from peripheral blood (PBL) and head kidney (HKL) to support FV3 infection was examined. RTG-2 supported a moderate level of FV3 replication while viral replication in RTL-W1 was minimal. The rest of the cell lines did not support viral replication but all succumbed to the infection and were killed by FV3. Lymphocyte-like cells from PBL and HKL were not killed by FV3 while macrophage-like cells were. Most of the cell lines died by an apoptosis-independent mechanism, presumably necrosis, while the monocyte/macrophage cell line, RTS11, died by an apoptosis-dependent mechanism. In addition, neoplastic macrophage-like human U937 cell line, and T lymphocyte-like PEER cell line were also infected with FV3 to compare their response to that of rainbow trout immune cells. U937 cells were killed by FV3 in an apoptosis-dependent manner; however, PEER T cells did not die from FV3 infection, a result similar to the lymphocyte-like fraction of rainbow trout PBL and HKL. In summary, most rainbow trout cell lines do not support significant FV3 replication; furthermore, cells of the lymphocyte origin appeared refractory to FV3 induced cell death while those of macrophage origin underwent apoptosis as a response to FV3. The last factor affecting the transmission of aquatic viruses examined in this thesis is the persistence of viruses in the aquatic environment. Virus persistence is influenced by natural environmental factors such as temperature, pH, desiccation and salinity, but the often unexplored anthropogenic factors can play a role. Therefore, the focus of this section was on the effect of one particular anthropogenic substance, Corexit 9500, on the infectivity of aquatic viruses with different physical characteristics. The effect of Corexit 9500, a dispersant used to clean up oil spills, on invertebrates, lower vertebrates, birds and human health have been examined but there is a significant lack of study on the effect of this dispersant on aquatic viruses. In this study, the effect of Corexit 9500 on four aquatic viruses of different structural composition was examined. Corexit 9500 reduced the titre of the enveloped viral hemorrhagic septicemia virus (VHSV) at all concentrations (10% to 0.001%) examined. The titre of frog virus 3 (FV3), a virus with both enveloped and non-enveloped virions, was only reduced at the high Corexit 9500 concentrations (10% to 0.1%). Corexit 9500 was unable to reduce the titre of non-enveloped infectious pancreatic necrosis virus (IPNV), but enhanced the titre of chum salmon reovirus (CSV) by 2-4 logs. With the ability to inactivate enveloped viruses and possibly enhance some non-enveloped viruses, Corexit 9500 has the potential to alter the aquatic virosphere.

29

Sansom, Bryan. "Toxicity Assessment of Oil Sands Process-Affected Water Using Fish Cell Lines". Thesis, 2010. http://hdl.handle.net/10012/5514.

Testo completo

Gli stili APA, Harvard, Vancouver, ISO e altri

Abstract (sommario):

Toxicity assessment of large numbers of oil sands process-affected waters (OSPW) are needed in order to reclaim mined oil sands aquatic reclamation scenarios, such as End Pit Lakes (EPLs). Conventional toxicity testing using whole animals can make this process extremely costly, thus alternatives are being sought. A non-lethal bioassay is being developed and validated to aid in supporting reclamation planning. This study employed six fish cell-lines (WF-2, GFSk-S1, RTL-W1, RTgill-W1, FHML, FHMT) in 24h viability assays for rapid fluorometric assessment of cellular integrity and functionality. Eight ml from forty-nine OSPW samples received from Syncrude Canada Ltd. were mixed with 2 ml of 5X concentrated L-15/ex minimal media solution and used to expose cells. After 24h exposure to OSPW samples, significant decreases in cell viability as measured by Alamar blue (AB) were seen in all cell lines for a number of samples. Bioassays were done in blind, but when OSPW chemical composition was revealed there was a consistent correlation between decreasing cell viability and increasing naphthenic acid (NA) concentrations present in the samples. Regression analysis yielded correlation coefficients2 as high as 0.6171 (WF-2 cell line, AB; p<0.0001). NAs have been identified as the chief toxicants in OSPW. Therefore, a fish-cell line bioassay sensitive to fluctuations in NA concentration could be a tool integral to the safe implementation and biomonitoring of wet reclamation landscapes in the Athabasca oil sands region, such as EPLs.

30

Vedor, Joana Sofia Martins. "Evaluation of the cytotoxicity of Asparagopsis armata exudate in fish cells". Master's thesis, 2021. http://hdl.handle.net/10773/30853.

Testo completo

Gli stili APA, Harvard, Vancouver, ISO e altri

Abstract (sommario):

The oceans present an enormous importance on our planet, representing almost 99% of the planet's living space. Its vastness gives the illusion of resistance to anthropogenic activity and infinite resources that has led to inappropriate exploitation over the years. A consequence of anthropogenic pressures the introduction of non-native species poses a devastating threat to biodiversity. The red macroalgae Asparagopsis armata, originally from Western Australia, is now distributed all over the planet and is abundant along the Portuguese coast. This species represents a threat to native species since it produces potentially toxic exudates, becoming highly invasive, predatorless and with high growth rates. The main objective of this study was to evaluate the toxicity of Asparagopsis armata exudates, using in vitro assays. Therefore, its cytotoxicity was evaluated for a cell line of gilthead sea bream (Sparus aurata), a fish species of high commercial value at national and European level. Assays were performed for 24 h, where cell viability was assessed, using the MTT and Resazurin reduction assay, and biochemical responses associated with antioxidant activity and biotransformation were evaluated after 24 h exposure. Overall, the data revealed that cell viability of gilthead seabream fish cells is significantly reduced when exposed to more than 25% A. armata exudate. It also induces an increase in non-protein thiol activity, indicative of an increased non-enzymatic antioxidant capacity in response to toxic compounds present in the exudate.
Os oceanos apresentam uma enorme importância no nosso planeta, representando quase 99% do espaço vital do planeta. A sua vastidão dá a ilusão de resistência à atividade antropogénica e recursos infinitos que tem levado à exploração imprópria ao longo dos anos. Consequência das pressões antropogénicas a introdução de espécies não-nativas representa uma ameaça devastadora à biodiversidade. A macroalga vermelha Asparagopsis armata, originária da Austrália Ocidental, está atualmente distribuída por todo o planeta sendo abundante na costa Portuguesa. Esta espécie representa uma ameaça para as espécies nativas dado que produz exsudados potencialmente tóxicos tornando-se altamente invasiva, sem predadores e com taxas de crescimento elevadas. O presente trabalho teve como objetivo principal a avaliação da toxicidade do exsudado da Asparagopsis armata, recorrendo a ensaios in vitro. Assim, foi avaliada a sua citotoxicidade para uma linha celular de dourada (Sparus aurata), uma espécie de peixe de elevado valor comercial a nível nacional e Europeu. Foram realizados ensaios de 24 h, onde foi avaliada a viabilidade celular, recorrendo ao ensaio de redução de MTT e Resazurina, e avaliadas respostas bioquímicas associadas à atividade antioxidante e de biotransformação após exposição de 24 h. De uma forma geral, os dados revelaram que a viabilidade celular das células de dourada é significativamente reduzida quando expostas a mais de 25% de exsudato de A. armata. Para além de que induz um aumento na atividade de tióis não proteicos, indicativo de um aumento da capacidade antioxidante não enzimática em resposta aos compostos tóxicos presentes no exsudato.
Mestrado em Biologia Molecular e Celular

31

El-Sweisi, Wail. "Evaluating the toxicity of eight reactive environmental contaminants by monitoring three measures of cell viability in two fish cell lines". Thesis, 2009. http://hdl.handle.net/10012/4930.

Testo completo

Gli stili APA, Harvard, Vancouver, ISO e altri

Abstract (sommario):

As fish cell cultures continue to be explored as alternatives to whole fish for evaluating the toxicity of environment chemicals, technical issues have emerged that influence results and thus need to be understood and standardized. These include carrier solvents, dosing protocols, exposure vessel, exposure media, viability endpoints, and cell lines. Some of these factors have been explored in this thesis for eight reactive contaminants exhibiting varied physicochemical properties using the rainbow trout cell lines RTgill-W1 and RTL-W1. Sodium dodecyl sulphate (SDS) was used as a reference (control) chemical. Cell viability was evaluated with alamar Blue, carboxyfluoroscein diacetate acetoxymethyl ester and neutral red as measures respectively of metabolic activity, plasma membrane integrity, and lysosomal function. Experimental in vitro EC50 values were compared to 1) pre-existing in vivo LC50s from the fathead minnow database and 2) pre-existing in vitro EC50s from the Halle database. Results point to good in vitro/in vivo correlations for menadione, dichlorophene, hexachlorophene, and acrolein. Poor correlations were observed for allyl alcohol, 4-fluoroaniline, acetaldehyde, and 2,3-dimethyl-1,3-butadiene due to a combination of solubility and volatility problems. Overall, the results suggest that the impact of different technical approaches on the evaluation of acute toxicity in vitro depends very much on the chemical class being investigated and less on the characteristics of the cell line. The in vitro cytotoxicity of reactive chemicals is challenging due to the nature of the chemicals’ physicochemical properties. Further improving the in vitro toxicity of reactive chemicals is a prerequisite for the ultimate goal of using fish cell cultures as acceptable, standard alternatives to the use of fish acute lethality assays.

32

Wang, Zheyu. "The effect of seal oil on paclitaxel induced cytotoxicity and apoptosis in breast carcinoma MCF-7 and MDA-231 cell lines /". 2004.

Cerca il testo completo

Gli stili APA, Harvard, Vancouver, ISO e altri

33

Heng-DaoLin e 林衡道. "Studies on betanodavirus non-structural protein B1 how to regulate the oxidative stresses in fish cell lines". Thesis, 2013. http://ndltd.ncl.edu.tw/handle/36483187060182673047.

Testo completo

Gli stili APA, Harvard, Vancouver, ISO e altri

Abstract (sommario):

碩士
國立成功大學
生物科技研究所碩博士班
101
As we known, some fish species are infected by betanodavirus such as grouper, which results in severe mortality and significant economic losses on the aquaculture industry. In previous studies, RGNNV can induce necrotic cell death via mitochondrial membrane (MMP) loss in grouper cells. The RGNNV B1 is belonging to early expression gene which plays a crucial role on anti-apoptotic function and reduces viral replication. But, whether B1 overexpression can regulate ROS is still unknown. And how to reduce cell death with virus infection is also unclear. In this study, we found that the B1 can induce superoxide anion (O2- ) production at 12h. And then the B1 can up-regulate some anti-oxidant enzymes such as Cu/Zn SOD, Mn SOD, catalase and arrest the cell cycle via oxidative stress response. On the other hand, the B1 can reduce cell death with treat H2O2, tBHQ (H2O2 inducer) and Act D (Actinomycin D), an apoptosis inducer. Furthermore, we still don’t know whether B1 protein targeting into nucleus is correlative to anti-death function. In the result, we found that B1 targeting into nucleus can correlate to B1 function. Finally, we analyzed the transcription level by cDNA microarray after EYFP-B1 transfection and try to find some hint for ROS and anti-cell death studies. In the result, the B1 protein may play a multifunction roles, which not only arrest cell cycle but also induce ROS production and cell death. This first finding may provide an insight into the molecular pathogenesis of betanodavirus infection in future. Keywords: Betanodavirus, nonstructural protein B1, reactive oxygen species (ROS), oxidative stress respone, anti-oxidant enzyme, cell cycle arrest, anti-apoptosis.

34

Polanská, Daniela. "Využití tkáňových linií pro toxikologii v životním prostředí". Master's thesis, 2020. http://www.nusl.cz/ntk/nusl-410429.

Testo completo

Gli stili APA, Harvard, Vancouver, ISO e altri

Abstract (sommario):

5 Abstract Five substances from the group of so-called personal care products, known for their low degradability and regular environmental detection, were tested for toxicity using two fish tissue lines (RTgill-W1 a RTG-2) isolated from rainbow trout (Oncorhynchus miykiss). The tested substances were hexadecylpyridinium chloride (HDP), chlorhexidine (CHX), octenidine (OCT), thymol (THM) and triclosan (TCS). A cell viability assay was performed with each of these compounds using Alamar Blue ™ (AB), 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM) and neutral red (NR) protocols. The results were used to construct dose-response curves along with an EC50 value for each of these substances. The EC50 values ranged from 0,51 (HDP) to 33,75 µg.ml-1 (THM) for RTgill-W1 and from 0,31 (HDP) to 33,37 µg.ml-1 (THM) for RTG-2. The theoretical LC50 estimation was calculated according to Tanneberger et al. (2013). For all substances, cytochrome P450 1A activity was monitored using 7-ethoxyresorufin-o-deethylase (EROD), four out of five tested chemicals were statistically positive for EROD, the highest EROD response was observed for the most toxic compound - HDP. Only TCS did not show statistically significant cytochrome P450 1A activity. In addition, oxidative stress was measured with the fluorescent dye...

35

Lin, Shiue-Lian, e 林學廉. "Establishment and Characterization of Ornamental Fish Cell Lines and Development of Methods for Detection of Koi Herpesvirus Disease". Thesis, 2014. http://ndltd.ncl.edu.tw/handle/83400287477881642317.

Testo completo

Gli stili APA, Harvard, Vancouver, ISO e altri

Abstract (sommario):

博士
國立臺灣大學
漁業科學研究所
102
Establishing ornamental fish cell lines is essential for researching the viral diseases that affect these ornamental fish, particularly ornamental fish species that can be artificially bred. Typically, because of high-density breeding and the flow of international trade, these fishes cause rapid viral disease spread and transmission, resulting in severe economic losses. This study reports the first successful cultivation of cell lines from the fin tissue of the marine ornamental fish species Apolemichthys trimaculatus, that is, the three-spot angelfish (TSAF). We successively cultivated 7 Carassius auratus or red carp (RC) cell lines from the fin (F), gill (G), heart (H), head kidney (HK), spleen (S), and ovum (O) tissues, yielding RCF, RCG, RCH, RCK, RCHK, RCS, and RCO cell lines. An RCHK macrophage (RCHKM) cell line was also cultivated from RC head kidney tissues. We also established 5 Cyprinus carpio (Koi) cell lines (KF, KG, KH, KB, and KO) from the fin, gill, heart, air bladder, and ovum tissues of koi. These cell lines can be continually reproduced and grown in simple cultivation conditions of 20°C-30°C and 10% FBS-L15. TSAF cell lines were highly susceptible to the eel herpesvirus in Formosa (EHVF), which ideally grows in 30°C environments, and to the infectious pancreatic necrosis virus (IPNV), endogenous viral elements (EVEs), and hippeastrum chlorotic ringspot virus (HCRV), which ideally grow in 18°C environments. Therefore, TSAF cell lines are extremely appropriate as virus detectors for cold and warm water fishes. The RCO, RCHKM, KF, KG, and KB cell lines were all susceptible to koi herpesvirus (KHV), generating the cytopathic effect commonly presented in herpesvirus. Hence, these cell lines can be used to mass-proliferate KHV. Accordingly, cell lines are the optimal tools for researching fish virology, pathology, molecular biology, and conducting environmental testing. A mutated KHV cell line, named NKV2, was identified at a koi breeding ground in Southern Taiwan. NKV2 cannot be detected using various KHV primers, carp interstitial nephritis and gill necrosis virus (CNGV) primers, thymidine kinase (TK) primers, and Gray primers, but can be detected using the carp pox cyprinid herpesvirus 1 (CyHV-1) helicase primer and CyHV-1 triplex primers, which are used to detect KHV. We designed and developed two new primers (i.e., the NKV2-2K and NKV2-1.5K primers) based on KHV primer. The proposed primers can be employed to detect both KHV and NKV2. An additional primer (i.e., the NKV2-TK primer) was designed and developed based on TK primer. This primer can be used to detect both KHV and NKV2 according to the introns of NKV2 are larger compared with those of KHV. The developed primers can be adopted in polymerase chain reaction (PCR) to detect KHV and NKV2 in the same time.

36

Fish, Jennifer [Verfasser]. "The evolution of neuronal progenitor cell division in mammals: the role of the abnormal spindle-like microcephaly associated (Aspm) protein and epithelial cell polarity / Jennifer Fish". 2007. http://d-nb.info/985849908/34.

Testo completo

Gli stili APA, Harvard, Vancouver, ISO e altri

37

Wong, Janice. "Use of fish cell lines to compare the cytotoxicity of Tetrabromobisphenol A with its degradation products and with an alternative brominated flame retardant". Thesis, 2011. http://hdl.handle.net/10012/6107.

Testo completo

Gli stili APA, Harvard, Vancouver, ISO e altri

Abstract (sommario):

Tetrabromobisphenol A, (TBBPA or Br4BPA), is a widely used brominated flame retardant (BFR). Although TBBPA and its breakdown products been found in river sediments, the environmental impact of their contamination is largely unknown. One breakdown product of TBBPA is bisphenol A (BPA), which has been studied intensively for its toxicology because it is used in the manufacturing of plastics and leaches from food containers, water bottles and pipes. Other breakdown products of TBBPA include tribromobisphenol A (Br3BPA), dibromobisphenol A (Br2BPA), and monobromobisphenol A (BrBPA) but little is known about their toxicology. Since TBBPA is toxic, there is a need to search for an alternative BFR, with one being tetrabromobisphenol A bis(2,3-dibromopropylether) or TBBPA-DBPE. However, almost nothing is known about the toxicology of this compound. Hence, two rainbow trout cell lines, RTL-W1 from liver and RTgill-W1 from gill, were used to evaluate the cellular toxicity of TBBPA, BPA, BrBPA, Br2BPA, Br3BPA and TBBPA-DBPE. The cells were exposed to these compounds for 24 h in the basal medium, L-15, to study their cytotoxicity and in L-15 with fetal bovine serum (FBS) to evaluate their capacity to induce 7-ethoxyresorufin o-deethylase (EROD) activity. Viability was measured with three fluorometric indicator dyes: Alamar Blue (AB) for metabolism, 5-carboxyfluorescein diacetate acetoxymethyl (CFDA AM) for cell membrane integrity, and Neutral Red (NR) for lysosomal activity. The concentrations causing a 50 % reduction in viability (EC50) as measured with these three dyes were used to compare the relative cytotoxicity of these chemicals. For both cell lines and with all viability endpoints, TBBPA was the most cytotoxic, with EC50s ranging from 2.33 to 3.11 ug/ml. BPA, BrBPA, Br2BPA, and Br3BPA also caused dose-dependent declines in cell viability but showed no consistent order of potency. None of the six compounds induced EROD activity, which suggests that they do not activate the aryl hydrocarbon receptor (AhR). Regardless of the endpoint or cell line, TBBPA-DBPE was not cytotoxic. This suggests that, from a toxicological perspective, this compound may be a suitable replacement for TBBPA as a BFR. BPA stood out from the other compounds in two regards. BPA caused a dose-dependent decline in cell viability for cultures in L-15 with FBS, whereas for the other compounds, little or no change in viability was seen in cultures with FBS. BPA elicited a decline in the ability of cells to reduce AB almost immediately upon its addition to cultures in a simple buffer, whereas as for other compounds a decline took time to develop. These results suggest that BPA exerts its cytotoxicity by a different mechanism different from the other compounds.

38

Yu, Wen-ya, e 余雯雅. "Using a mouse model to assess colon cancer stem-like cell performance and examine eliminate capacity following dietary supplementation with fish oil and selenium yeast". Thesis, 2014. http://ndltd.ncl.edu.tw/handle/fp879x.

Testo completo

Gli stili APA, Harvard, Vancouver, ISO e altri

Abstract (sommario):

碩士
國立臺灣海洋大學
食品科學系
102
Traditional cancer therapies are high efficiency to kill cancer cells, but some of the advanced malignancy cells can still survive. Recent studies have shown that the small population of tumor cells, known as cancer stem cells (CSCs). These CSCs have the ability to promote tumor drug-resistant and metastasis, and play an important role of malignant progression and recurrence. Colorectal cancer (CRC) is one of the common malignant neoplasm, that is difficult to discover in the early stage and often metastasizes to liver and lung. Omega-3 polyunsaturated fatty acids (PUFAs) derived from fish oil has been reported has the capability to inhibit tumor growth and metastasis. Selenium yeast is also able to suppress tumor invasion and anti-angiogenesis. However, whether the combination effects of fish oil and selenium yeast mitigate the adverse effects of CSCs is still unclear. First, we treat cells with chemotherapy drug (cisplatin) to characterize the drug resistant of CSCs in BALB/c mouse colon carcinoma cells (CT26). The results showed that the survival cells have higher chemo-resistance and CSCs-related gene expression. Therefore, we establish CT26 as a colon CSCs research model in the following experiments. We screen the side population cells (SP cells) in CT26 with Hoechst dye, which have better drug- resistance and higher CSCs-related gene expression. Furthermore, the migratory capacity and colony formation ability of SP cells are higher than non-population cells (NSP cells). In vivo, we demonstrate that SP cells have stronger tumorigenic ability, more body weight loss and lung metastasis. After analyses lung tissues, the gene expression of metastasis–associated chemokines and matrix metalloproteinases are increased. In further experiments, the dietary supplementation with fish oil and selenium yeast can attenuate body weight loss and lung metastasis. The expression of metastasis–associated chemokines and matrix metalloproteinases are suppressed. Thus, fish oil and selenium yeast supplementation can lower the possibility of cancer metastasis which caused by cancer stem cells.

39

Σαλαμαστράκης, Σπυρίδων. "Ανοσοσήμανση πρωτεϊνικών δομών (μικροσωληνίσκοι-κεντρόσωμα-κινητοχώρος) και in situ υβριδοποίηση με φθοροχρώματα σε κυτταρικές σειρές ανθρώπου και μυός επιβεβαιώνουν την ανευπλοειδογόνο δράση της φαρμακευτικής ένωσης υδροχλωροθειαζίδιο". 2006. http://nemertes.lis.upatras.gr/jspui/handle/10889/407.

Testo completo

Gli stili APA, Harvard, Vancouver, ISO e altri

Abstract (sommario):

Η ακεραιότητα και η λειτουργία της μιτωτικής συσκευής διαδραματίζει ουσιώδη ρόλο για τον ορθό προσανατολισμό και την ολίσθηση των χρωμοσωμάτων στους πόλους της ατράκτου, οδηγώντας στον ισομερή διαχωρισμό των χρωμοσωμάτων κατά τη μιτωτική ή μειωτική διαίρεση. Τροποποιήσεις του δικτύου των μικροσωληνίσκων (α- και β-τουμπουλίνη) και των κέντρων οργάνωσης αυτών (ΜΤΟC, γ-τουμπουλίνη) είναι δυνατόν να προκαλέσουν βλάβες στη μιτωτική συσκευή, διαταράσσοντας το χρωμοσωματικό αποχωρισμό, με αποτέλεσμα το σχηματισμό ανευπλοειδικών κυττάρων. Η διουρητική φαρμακευτική ένωση υδροχλωροθειαζίδιο (HCTZ) χορηγείται κατά της υπέρτασης και είναι γνωστό ότι προκαλεί μη αποχωρισμό σε διπλοειδή στελέχη του μύκητα Aspergillus nidulans. Πρόσφατες μελέτες της ερευνητικής μας ομάδας έχουν δείξει ότι επάγει αυξημένες συχνότητες μικροπυρήνων και διαταράσσει το χρωμοσωματικό αποχωρισμό σε καλλιέργειες ανθρωπίνων λεμφοκυττάρων in vitro. Στην παρούσα εργασία μελετήθηκε η επίδραση του HCTZ στην οργάνωση του δικτύου των μικροσωληνίσκων κατά τη μεσόφαση και τη μίτωση, με συνδυασμένη εφαρμογή μεθόδων διπλής ανοσοσήμανσης των πρωτεϊνών των μικροσωληνίσκων, του κεντροσώματος και του κινητοχώρου. Εφαρμόστηκε επίσης in situ υβριδοποίηση με φθοροχρώματα (FISH), με α-satellite πανκεντρομερικό ανιχνευτή, για τον εντοπισμό μη ενσωματωμένου (lagging) χρωμοσωματικού υλικού. Η μελέτη πραγματοποιήθηκε σε κυτταρικές σειρές μυός C2C12 και ανθρώπου HFFF2. Παρατηρήθηκε ότι το HCTZ προκαλεί μείωση του ρυθμού διαίρεσης, αποδιοργάνωση του δικτύου των μικροσωληνίσκων και αυξημένη συχνότητα ανώμαλων μεταφάσεων με ποικίλο αριθμό σημάτων γ-τουμπουλίνης. Αυξάνει το ποσοστό των μεταφάσεων και μειώνει το ποσοστό των ανα-τελοφάσεων, προκαλώντας συσσώρευση των κυττάρων στο στάδιο της μετάφασης. Επίσης, επάγει τη χρωμοσωματική καθυστέρηση, καθώς αυξάνει τη συχνότητα των μικροπυρήνων που παρουσιάζουν τόσο σήμα κινητοχώρου όσο και σήμα κεντρομέρους. Η γενετική δράση του στα κύτταρα C2C12 δε φαίνεται να επηρεάζεται σημαντικά από τη χρονική διάρκεια έκθεσης των κυττάρων σ’ αυτό. Από τις δυο κυτταρικές σειρές που χρησιμοποιήθηκαν φαίνεται ότι τα κύτταρα C2C12 είναι περισσότερο ευαίσθητα στην απόκριση στο HCTZ. Τα αποτελέσματα μας, υποδεικνύουν ανευπλοειδογόνο δράση της φαρμακευτικής ένωσης, επιβεβαιώνοντας και ενισχύοντας προηγούμενα ευρήματα της ερευνητικής μας ομάδας.
The integrity and function of mitotic apparatus play essential role for the equitable orientation and the slipping of chromosomes to the spindle poles, indicating normal distribution of chromosomes during mitotic or meiotic division. Modifications of the microtubule network (α- and β- tubulin) and microtubule-organizing centers (MTOC, γ- tubulin) may cause severe damage to the mitotic apparatus, disturbing the segregation of chromosomes and resulting to aneuploid cells. The diuretic drug hydrochlorothiazide (HCTZ) is used for the treatment of hypertension and has been found to induce non-disjunction in diploid strains of Aspergillus nidulans. Recent studies of our team have shown that HCTZ produces increased frequencies of micronuclei and disturbs chromosome segregation in human lymphocytes cultures treated in vitro. In the present study was investigated the effect of HCTZ on the organization of the microtubule network during mesophase and mitosis, with combined application of double immunofluorescence staining assay, for the visualization of microtubules, centrosomes and kinetochore proteins. Fluorescence in situ hybridization (FISH), with α-satellite pancentromeric probe, was also applied, for the localization of not integrated (lagging) nuclear material. The study was realized in C2C12 mouse cells and HFFF2 human cells. Our results revealed that HCTZ causes decrease of the cell division rate, disorganization of the microtubule network and increased frequency of abnormal metaphases with various γ-tubulin signals. HCTZ increases the percentage of metaphases and decreases the percentage of ana-telophases, indicating a metaphase arrest. Also, induces chromosome delay, as was shown from the high frequency of micronuclei that presents kinetochore and centromere signal. The genetic activity of HCTZ in C2C12 cells does not appear to be significantly influenced by the duration of the cell’s exposure time to the drug. It appears that C2C12 mouse cells are more sensitive in their response to the HCTZ. These results indicate aneugenic activity of this drug, confirming and enhancing our previous findings.

Tesi sul tema "Fish cell line"

Cita una fonte nei formati APA, MLA, Chicago, Harvard e in molti altri stili