Dissertations / Theses on the topic 'RoGFP2'

Les stérilités mâles cytoplasmiques (SMC) résultent d'une incompatibilité nucléo-cytoplasmique. Le cytoplasme (presque toujours la mitochondrie) peut porter un gène de stérilité mâle, et le noyau peut restaurer la fertilité pollinique ou non. Les mécanismes physiologiques conduisant à la mort pollinique restent largement incompris. Plusieurs hypothèses ont été proposées, parmi lesquelles une déficience en ATP. Une SMC gamétophytique a été découverte chez A. thaliana. Une phase ouverte de lecture codant possiblement un peptide de 117 acides aminés, appelée orf117Sha, a été identifiée comme facteur de stérilité candidat.Au cours de ma thèse, j'ai cherché à valider le rôle de l'orf117Sha, et à comprendre comment une anomalie mitochondriale pouvait induire cette SMC. Aucune différence n'a pu être détectée au niveau de l'ARNm de l'orf117Sha entre les lignées stérile et restaurée, mais sa protéine semble accumulée uniquement dans la lignée stérile. La phénocopie par transgénèse de la SMC a suggéré un effet délétère de l'ORF117SHA dans les gamétophytes mâle et femelle.La description cytologique de la SMC montre une mort pollinique progressive à partir du stade binucléé. Auparavant, les mitochondries du pollen gonflent puis éclatent, et le développement s'arrête. L’utilisation de senseurs génétiquement encodés mesurant la concentration en ATP (ATeam) et l'état redox du glutathion (roGFP2-Grx1) a permis la mesure de ces facteurs en microscopie confocale, dans des tissus végétatifs et dans le pollen. La production d'ATP ne semble pas affectée dans la lignée stérile, contredisant l'hypothèse de l'ATP. Le glutathion mitochondrial est suroxydé dans la lignée stérile, à la fois dans les tissus végétatifs étudiés et le pollen, qui serait liée à la SMC car annulée par la restauration génétique de fertilité.Avec cette étude, j'apporte des arguments en faveur de l'orf117Sha dans l'induction de la SMC Sha, et je décris les évènements préalables à l'avortement du grain de pollen. Mes résultats permettent de mieux comprendre les évènements physiologiques conduisant à la mort du pollen
This work aims at better understand the events leading to pollen abortion in a recently discovered gametophytic cytoplasmic male sterility (CMS) in Arabidopsis thaliana. Although CMS have been widely used in hybrid seed production in many crops, the physiological mechanisms leading to pollen death by the mitochondrial sterilizing genes in the permissive (maintainer) nuclear backgrounds are poorly understood. Association genetics previously identified orf117Sha as a candidate mitochondrial CMS-associated gene.In a first part, I analyzed the expression of the orf117Sha gene in sterile plants and in fertile plants carrying nuclear genes restoring male fertility. I observed unusual features of its mRNA, but detected no difference at this level between sterile and restored plants. Oppositely, the ORF117SHA protein seems to be accumulated specifically in the sterile line, supporting its role in CMS. A phenocopy attempt by transgenesis suggested a possible link between a female and male gametophytic lethality and the ORF117SHA, even though few individuals could be analyzed.In a second part, I observed pollen development in sterile plants and fertile controls using different cytological approaches. My results show a progressive pollen death starting from the binucleate stage in the sterile. Prior to abortion, pollen mitochondria swell before rupture, and the development stops. I used confocal microscopy combined with genetically encoded sensors to explore specific physiological features in pollen and vegetative tissues of sterile plants. With ATeam, which allows the assessment of ATP content in the cytosol, I could challenge the generally accepted hypothesis of an ATP deficiency leading to pollen abortion in CMS. Indeed, the ATP production does not seem to be affected in the sterile line. With a mitochondria-addressed roGFP2-Grx1, I was able to assess the redox state of the glutathione pool in vegetative tissues and in the male gametophyte. I observed an overoxydation of the glutathione pool in mitochondria of the sterile line, in vegetative tissue investigated and in the pollen grain. This overoxydation seems to be linked to the CMS as it is annihilated by the presence of restorer genes.My results pave the way for further exploration of the links between the sterility protein, mitochondrial morphology changes, mitochondrial overoxydation, and pollen development arrest and death in the A. thaliana CMS

Au cours des dix dernières années, le développement de biosenseurs redox fluorescents a permis de générer des outils permettant d'étudier les dynamiques in vivo de molécules comme les formes réduite et oxydée du glutathion ou le peroxyde d'hydrogène. La cystéine étant un métabolite clé du métabolisme du soufre, l'objectif de ce projet de thèse était de développer un biosenseur redox fluorescent spécifique de la cystéine en couplant une oxydoréductase à la roGFP2 (reduction-oxidation green fluorescent protein). Tout d'abord les activités de plusieurs isoformes de cystéine désulfurases (CD) et des protéines à domaine rhodanese (Rhd), catalysant respectivement la désulfuration de la cystéine et des réactions de trans-persulfuration ont été analysées in vitro afin de déterminer si elles pouvaient constituer de bons candidats pour cette activité oxydoréductase. Ces analyses ont mis en évidence qu'une protéine chimérique naturelle bactérienne possédant des domaines CD et Rhd oxyde efficacement la roGFP2, au travers de réactions de trans-persulfuration depuis la cystéine vers la roGFP2. Cette protéine candidate a ensuite été fusionnée à la roGFP2 pour générer le biosenseur CD-Rhd-roGFP2. In vitro, cette protéine est sensible à l'oxydation en présence de concentrations physiologiques en cystéine alors que l'oxydation par le thiosulfate, autre substrat potentiel du domaine Rhd, est négligeable. D'une part, les réactions de trans-persulfuration entre les domaines protéiques menant à l'oxydation de la roGFP2 ne sont pas ou très peu inhibées par les systèmes réducteurs physiologiques. Néanmoins, le système glutathion-glutarédoxine réduit spécifiquement la roGFP2. L'expression de ce biosenseur chez la bactérie Escherichia coli, a révélé une réponse dynamique en réponse à des ajouts exogènes de cystéine ou de cystine ouvrant la voie à des études similaires dans les organites d'autres organismes modèles eucaryotes
Over the last decade, the development of fluorescent redox biosensors has provided tools to study the in vivo dynamics of molecules such as the reduced and oxidized forms of glutathione or hydrogen peroxide. Cysteine being a key metabolite of sulfur metabolism, this PhD project aimed at developing a fluorescent redox biosensor specific for cysteine by coupling an oxidoreductase to roGFP2 (reduction-oxidation green fluorescent protein). First, the activities of several isoforms of cysteine desulfurases (CD) and rhodanese-domain containing proteins (Rhd), catalyzing cysteine desulfuration and trans-persufidation reactions, respectively, were analyzed in vitro in order to determine whether they could constitute good candidates for this oxidoreductase activity. These analyses revealed that a natural chimeric protein possessing both CD and Rhd domains efficiently oxidizes roGFP2, by catalyzing trans-persulfidation reactions from cysteine to roGFP2. This candidate protein was then fused to roGFP2 to generate the CD-Rhd-roGFP2 biosensor. In vitro, this protein is sensitive to oxidation in the presence of physiological concentrations of cysteine whereas oxidation by thiosulfate, another potential substrate of the Rhd domain, is negligible. In addition, the trans-persulfidation reactions between the protein domains leading to the oxidation of roGFP2 are not inhibited by physiological reducing systems. Nevertheless, the glutathione/glutaredoxin system specifically reduces roGFP2. The expression of this biosensor in the bacterium Escherichia coli revealed a dynamic response of the biosensor to exogenous addition of cysteine or cystine, paving the way for similar studies in organelles from other eukaryotic model organisms

Consensus opinion suggests that apoptosis occurs when the intracellular redox potential reaches its oxidative range, i.e. when the balance between oxidants and reductants is disturbed. An understanding of the links between redox potential and the induction of apoptosis in cells could improve our understanding of the process and help to predict therapeutic responses. This study investigates the changes in redox potential at distinct stages of apoptosis induced in the human cervical cancer cell line, HeLa. Stages of the apoptotic process were defined by loss of mitochondrial membrane polarisation (ΔΨm), membrane phosphatidyl serine exposure, caspase-3 activation, and nuclear fragmentation. To measure real-time redox potential change in apoptotic cells two independent methods were used: (1) expression of redox-responsive green fluorescent protein (roGFP2) measured by flow cytometry and (2) redox-responsive nanosensors detected by surface enhanced Raman spectroscopy (SERS). roGFP2 measurements showed that HeLa cells demonstrate a shift towards an oxidative redox state during the later stages of apoptosis and this was preceded by loss of ΔΨm. The relationship between these two events was investigated by transient inhibition of mitochondrial permeability transition pore opening using the inhibitor bongkrekic acid (BKA) pre-treatment. At the cell population level, transient exclusion of the mitochondrial contribution delayed two key events of apoptosis in the first two hours measured by nuclear fragmentation and loss of ΔΨm. However, BKA treatment did not affect redox potential, reported by roGFP2, when compared with controls. Therefore, this suggests that mitochondria do not contribute towards the overall redox potential change in apoptosis. To gain insight into the significance of redox change at the earliest stages of apoptosis, single cell studies were performed. SERS, employing simultaneous redox potential and intracellular pH measurements using two synthetic nanosensors AQ-NS and MBA-NS, showed that BKA pre-treatment resulted in increased alkalinity and the cells were consequently protected from induction of apoptosis in the first thirty minutes of the kinase inhibitor staurosporine treatment. Measurements with SERS nanosensors allowed for adjustment for pH, which provides a clearer insight into redox potential dynamics, with consideration of the environment, and accurate quantitative assessment of redox at early stages of apoptosis. Together these data suggest that while roGFP2 is a valid method to use at a population level, SERS is a more sensitive method for measuring the redox potential of the cell at the early stages of apoptosis.

O estresse oxidativo e a enzima NADPH oxidase-2 (NOX2) estão associados com a diminuição da massa funcional de células-β em pacientes com diabetes do tipo 2 (DT2). Neste estudo, testamos o papel da NOX2 sobre a glicotoxicidade em células-β. Ilhotas de camundongo C57BL/6J nocautes ou não para NOX2 (NOX2-KO e WT, respectivamente) foram isoladas e cultivadas por até 3 semanas em 10 ou 30 mmol/l de glucose (G10 e G30, respectivamente). A secreção de insulina foi maior nas ilhotas NOX2-KO vs. WT sem apresentar diferenças metabólicas ou do potencial redox da glutationa citosólica (EGSH). O cultivo de ilhotas em G30 aumenta a concentração de H2O2 e a oxidação de tióis no compartimento citosólico, seguido por aumento de apoptose de células-β, mas, preservando a reposta máxima secretória. Estas respostas foram quase idênticas em ambos os tipos de ilhotas. Em conclusão, a NOX2 regula negativamente a secreção de insulina em ilhotas de camundongos C57BL/6J, mas não é um componente crítico para a sobrevivência de células β em um modelo in vitro de glicotoxicidade.
Oxidative stress and NADPH oxidase-2 (NOX2) enzyme are associated to the decline of the functional β-cell mass in type 2 diabetes (T2D). Here, we tested the role of NOX2 on β-cell glucotoxicity. NOX2 knockout (NOX2 KO) and wild type (WT) C57BL/6J mice islets were isolated and cultured up to 3 weeks at 10 or 30 mmol/l glucose concentrations (G10 and G30, respectively). The insulin secretion was higher in NOX2-KO vs. WT islets despite similar metabolic and cytosolic glutathione-redox potential (EGSH) changes. The prolonged culture at G30 increases the H2O2 concentration and cytosolic thiol oxidation, followed by increased βcell apoptosis but preserving maximal secretory response. These responses were almost identical in both types of islets. In conclusion, NOX2 is a negative regulator of insulin secretion in C57BL/6J mouse islets, but is not a critical component for β-cell survival in a model of glucotoxicity in vitro.

Veränderungen im zellulären Oxidationszustand durch reaktive Sauerstoffspezies (ROS) sind in biochemischen Signalwegen, aber auch an der Entstehung von neurodegenerativen und anderen neuropathologischen Krankheiten beteiligt. Genetisch kodierte Redoxfarbstoffe haben sich in jüngerer Zeit als deutlich überlegen gegenüber der herkömmlichen Redox-Bildgebung gezeigt, um auch dynamische und quantitative Messungen des Redox-Status in komplexeren und adulten Organismen durchführen zu können. In dieser Arbeit wurde ein neues transgenes Mausmodell mit zytosolischer Expression des optischen Redox-Indikator roGFP1 in Neuronen unter der Kontrolle des Thy1-Promotors (C57BL/6-TG(Thy1.2-roGFP1c)) eingehend charakterisiert. Die transgenen Tiere zeigen einen mit verschiedenen Verhaltenstest und der Überprüfung verschiedener relevanter physiologischer Parameter sichergestellten gesunden Phänotyp. Bei an akuten Hirnschnitten und formalinfixierten Proben durchgeführten Messungen mit 2-Photonen-Laser-Scanning-Mikroskopie und herkömmlicher Fluoreszenzmikoskopie konnte eine kräftige Expression von roGFP1 vor allem in der CA1-Region, aber auch cortikal sowie im Hirnstamm gefunden werden. Durch Gegenfärbung mit fluoreszenmarkierten anti-NeuN-Antikörpern wurde eine extraneuronale Expression im Gehirn ausgeschlossen. Die Baseline der roGFP1-Antwort und Veränderungen im Oxidationszustand durch Inkubation von akuten Hirnschnitten mit Oxidations- und Reduktionsmitteln und unter Anoxie wurden dynamisch in Echtzeit mittels Fluoreszenzmikroskopie im Hippokampus und Neocortex bestimmt. Daraus kann geschlossen werden, dass das untersuchte C57BL/6-TG(Thy1.2-roGFP1c) Mausmodell funktionelle Redox-Indikatoren in ausreichendem Maße in großen Teilen des Gehirns exprimiert. Die Verfügbarkeit dieser Mäuse sollte sich als wichtig für das tiefergehende Verständnis der Rolle reaktiver Sauerstoffspezies und damit einhergehender Veränderungen des Redox-Status sowohl in der physiologischen Kontrolle der Zellfunktion als auch in neuropathologischen Prozessen erweisen.

Parkinson’s disease is a neurodegenerative disorder characterized by a loss of dopaminergic neurons, where mitochondrial dysfunction and neuroinflammation are implicated in this process. However, the exact mechanisms of mitochondrial dysfunction, oxidative stress and neuroinflammation leading to the onset and development of Parkinson’s disease are not well understood. There is a lack of tools necessary to dissect these mechanisms, therefore we engineered genetically encoded fluorescent biosensors to monitor redox status and an inflammatory signal peptide with high spatiotemporal resolution. To measure intracellular redox dynamics, we developed red-shifted redox sensors and demonstrated their application in dual compartment imaging to study cross compartmental redox dynamics in live cells. To monitor extracellular inflammatory events, we developed a family of spectrally diverse genetically encoded fluorescent biosensors for the inflammatory mediator peptide, bradykinin. At the organismal level, we characterized the locomotor effects of mitochondrial toxicant-induced dopaminergic disruption in a zebrafish animal model and evaluated a behavioral assay as a method to screen for dopaminergic dysfunction. Pairing our intracellular redox sensors and our extracellular bradykinin sensors in a Parkinson’s disease animal model, such as a zebrafish toxicant-induced model will prove useful for dissecting the role of mitochondrial dysfunction and inflammation in Parkinson’s disease.