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.

This master’s thesis contains a description of mesenchymal stem cells (MSC). The work includes knowledge about the process of migration and differentiation of MSCs. The theoretical part of this thesis also deals with the problem of cell cultivation in 2D and 3D environments. The most used materials for creating 3D scaffolds are described here. The practical part contains the description of the cell culture protocol for passaging and also describes the experiment in the cell laboratory. The results of the experiment are discussed using confocal microscope images. The proposed experiment was tested with a sufficient number of repetitions. The processing of microscopic images was performed in the MATLAB programming environment.

The theoretical part of this master's thesis is focused on research of confocal microscopy and FLIM method. There are a principles and types of confocal microscopy and the use of broad-spectrum laser as a basic light source of these microscopes. It gives what the FLIM method and its use not only in cell biology. The practical part thesis includes the acquisition of three sets of fluorescence intensity images with use of applications tunable pulsed laser, function TimeGate and detection of hybrid detectors. For practical elaboration of this thesis is in the software Fiji created a plugin, which is the source code in the Java programming language. The types of plugins and their uses are described in the third chapter of the thesis. This plugin including the graphical user interface in the form of the dialog box, proceses the fluorescence intensity images and creates a graphical representation of data showing the fluorescence lifetime, so called pseudocolor map.

La gonococcie est une infection sexuellement transmissible due au gonocoque. Elle est devenue un problème majeur de santé publique du fait de la multirésistance aux antibiotiques, mais surtout de la résistance au traitement de dernière intention actuellement en vigueur. Lactobacillus crispatus, une bactérie naturelle, commensale du vagin de la femme, s’est montré efficace pour inhiber le gonocoque. Les gels, une forme galénique bien acceptée, sont déjà utilisés pour le traitement des infections genitales de la femme. Disposer d’un gel contenant Lactobacillus crispatus, efficace, facile à administrer par la femme elle-même parait donc attractif pour la prévention de la gonococcie. Ainsi, nous avons conçu un gel à base d’un polymère thermogélifiant, le poloxamer 407 et d’un polymère biocompatible, l’alginate de sodium. Dans un premier temps, une étude physico-chimique du mélange de polymères a permis de retenir les concentrations optimisées. Dans un second temps, la souche de Lactobacillus choisie a été caractérisée et introduite dans le mélange de polymères. Les propriétés physicochimiques dont les caractéristiques rhéologiques, l’expulsion d’un dispositif d’administration, la stabilité, la microstructure ainsi que l’efficacité in vitro du gel obtenu ont été étudiés. Une répartition homogène de Lactobacillus crispatus a été observée dans le gel. Ce système est facilement administrable et possède des propriétés rhéologiques favorables à son étalement et son maintien dans la lumière vaginale. Ce gel a permis d’inhiber la croissance du gonocoque in vitro. / Gonorrhea is a sexually transmitted infection caused by Neisseria gonorrhoeae. It has become a major public health issue due to multidrug resistance, especially resistance to the current last-intention treatment.Lactobacillus crispatus, a natural bacterium, commensal to the woman's vagina, has been shown to inhibit Neisseria gonorrhoeae. Gels, a well-accepted dosage form, are already used for the treatment of woman's genital infections. Having a gel containing Lactobacillus crispatus, that is effective, easy to administer by the woman herself, would be ideal for the prevention of gonorrhea. Thus, we designed a gel based on a thermogelling polymer, poloxamer 407, and a biocompatible polymer, sodium alginate. First, a physicochemical study of the polymers mixtures allowed to select the optimized concentrations. Second, the selected Lactobacillus strain was characterized and introduced into the optimized polymer mixture. Physicochemical properties including rheological characteristics, expulsion from a device, stability, microstructure as well as in vitro gel efficacy were studied. A homogeneous distribution of Lactobacillus crispatus was observed in the gel. It was easily administered and its rheological properties were suitable for its spreading and its long reidence time in the vaginal lumen. This gel showed an inhibition of gonococcal growth in vitro.

Background: Assessment of platelet vitality is important for patients presenting with inherited or acquired disorders of platelet function and for quality assessment of platelet concentrates. Methods: Herein we combined live stains with intra-vital confocal fluorescence microscopy in order to obtain an imaging method that allows fast and accurate assessment of platelet vitality. Three fluorescent dyes, FITC-coupled wheat germ agglutinin (WGA), tetramethylrhodamine methyl ester perchlorate (TMRM) and acetoxymethylester (Rhod-2), were used to assess platelet morphology, mitochondrial activity and intra-platelet calcium levels. Microscopy was performed with a microlens-enhanced Nipkow spinning disk-based system allowing live confocal imaging. Results: Comparison of ten samples of donor platelets collected before apheresis and platelets collected on days 5 and 7 of storage showed an increase in the percentage of Rhod-2positive platelets from 3.6 to 47 and finally to 71%. Mitochondrial potential was demonstrated in 95.4% of donor platelets and in 92.5% of platelets stored for 7 days. Conclusion: Such fast and accurate visualization of known key parameters of platelet function could be of relevance for studies addressing the quality of platelets after storage and additional manipulation, such as pathogen inactivation, as well as for the analysis of inherited platelet function disorders. / Hintergrund: Die Vitalitätsbestimmung von Blutplättchen ist sowohl für die Analyse angeborener Plättchendefekte als auch für die Qualitätsbestimmung von Plättchenkonzentraten von zentraler Bedeutung. Methoden: In der vorliegenden Arbeit stellen wir eine Methode vor, die mittels einer Kombination von Vitalfarbstoffen und konfokaler «Real time»-Mikroskopie neue Einblicke in die Vitalitätsbestimmung lebender Plättchen ermöglicht. Mittels der Zugabe von FITC-gekoppeltem Weizenkeimlektin (WGA), Tetramethylrhodamin-Methylesterperchlorat (TMRM) und Acetoxymethylester (Rhod-2) wurde bei lebenden Blutplättchen deren Morphologie, mitochondriale Aktivität und Veränderungen im Calcium-Haushalt im Rahmen der Lagerung analysiert. Für die Mikroskopie wurde ein Nipkow-System gewählt, das eine konfokale Mikroskopie lebender Zellen ermöglicht. Ergebnisse: Der Vergleich von 10 humanen Blutplättchenproben zu Beginn bzw. nach 5 und 7 Tagen Lagerung zeigte einen Anstieg der Rhod-2-positiven Plättchen von 3,6 über 47 auf 71%. Die Anzahl der Blutplättchen mit TMRM-positiven Mitochondrien hingegen lag vor der Lagerung bei 95,4% und nach den 7 Tagen Lagerung bei 92,5%. Schlussfolgerung: Die hier vorgestellte Methodik der Bildgebung zur Bestimmung vitaler Parameter von Blutplättchen eignet sich als ergänzende Analysemodalität für eine bessere Bestimmung der Blutplättchenqualität. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

La photo-isomérisation de l’azopolymère cause des mouvements de masse permettant d'inscrire des figures d'interférences à la surface de films minces. Les films sont répliqués sur un substrat en PDMS transparente t étirable. Il forme des réseaux de phase. Les figures de diffraction décrivent les surfaces, elles sont utilisées pour modéliser numériquement les réseaux, quelles que soient les contraintes.Le projet WOLF vise à fabriquer des nano lasers blancs à colorants organiques. Un montage complexe de caractérisation est développé. Le module d’excitation permet d’illuminer les nanotubes un à un. Le pompage tente de maximiser l’émission laser d’un unique nano objet. La collection du signal repose pricipalement sur l’utilisation d’une fibre optique ouvrant la porte de la microscopie confocale. La spectroscopie associée au montage révèle la formation de cavités au sein des nano bâtonnets organiques. Malgré la faible puissance du signal, les modes sont étudiés,mettant en évidence des nano-cavités. Les nano-objets sont parfois étudiés dans des liquide. La micro-fluidique est développée au laboratoire. Sans salle blanche une méthode photolithographique est développée à bas coûts. Les puces nouvellement créées sont testées dans le mélange de deux fluides et lors de la caractérisation de nanoparticules en suspension par leur mouvement Brownien. Cette thèse à permis de mettre en place les outils d’observation de nano-objets uniques / Azopolymer photo-isomerization causes matter motion. This phenomena is responsible for the inscription of the interference pattern on surface of thin films. The films are replicated by pouring PDMS on it. PDMS is a transparent and stretchable polymer. The diffraction pattern of these gratings describes the surface. These informations are used to numerically simulate the surface irrespective of the mechanical constraints.The WOLF project try to synthetize white lightorganic nanolasers. For that purpose, a complex setup has been realised. The excitation part of the setup illuminates a single nano-laser or a few. The pump source is designed to optimise the amplified emission of the nanolasers. The collection of the signal is based on an optical fiber.This fibre has given us the chance to use confocal microscopy. Spectroscopy combined with our setup show the existence of nanocavities among nanotubes. In spite of the low signal power, the emitting modes are studied and show the cavity effect of these lasers. In another context, nano-objets are sometimes studied in solution. For that reason, a new experiment has been setup, namely, microfluidic. Without the use of a clean room, a low cost photolithograpic method is developped. The microfluidic chips are tested during the mixing of to liquids and during the obsevation of the brownian motion of particules. This PhD thesis has given the tools needed for the characterization of single nano-objects.

<p> The plant cytoskeleton is commonly considered a vital component of cell growth and development; however, it also plays a critical role in plant immunity. During plant immunity, the cytoskeleton orchestrates rapid and precise immune-associated processes. For instance, the cytoskeleton mobilizes and orients the movement of organelles, proteins, and chemical signaling. To counter plant immunity, bacterial pathogens deliver virulence proteins, known as T3Es (type III effectors), into plant cells through a needle-like apparatus called the type III secretion system (T3SS). A novel T3E, called RipU, interacts with the cytoskeleton. Data has shown that RipU co-localizes with cytoskeletal markers in tobacco leaves. Ectopic expression of RipU can suppress PTI responses like ROS bursts or seedling growth inhibition. Tomato plants inoculated with <em>Rs</em> K60 lacking RipU showed less wilting and root colonization, suggesting that RipU plays a role in pathogenesis and virulence. Furthermore, inducible expression of RipU in Arabidopsis dramatically alters plant development. These plants have wavy roots, branching root hairs, and underdeveloped true leaves. Our results suggest that by targeting the cytoskeleton, RipU contributes to <em>Rs</em> K60s pathogenicity and virulence. </p>

Secretory phospholipase A2 hydrolyzes phospholipids at a lipid-water interface, resulting in pro-inflammatory products being released from cell membranes. Healthy cells are resistant to cleavage by this enzyme, but apoptotic cells become susceptible to its activity. Only bilayers with certain characteristics are able to be hydrolyzed. Most recently, studies in this lab have emphasized the idea that the biophysical state of the bilayer (in terms of lipid order, spacing, and fluidity) is relevant in determining the probability of one phospholipid escaping the membrane to be hydrolyzed. Prior to this study, it had been shown that apoptotic cells undergo biophysical alterations that weaken inter-lipid interactions early in apoptosis. The purpose of this dissertation was to examine these changes in more detail, define them more clearly on the molecular level, and suggest possible mechanisms responsible for their occurrence. First, the role of increased membrane permeability in susceptibility to the phospholipase was investigated. S49 cells were treated with ionomycin or apoptotic agents and assayed for merocyanine 540 staining of the membrane and membrane permeability to a vital dye. Human group X and snake venom isoforms were active towards all treated cells, but human groups V and IIa only hydrolyzed cells that were moderately permeable to the vital dye. Different isoforms must then be sensitive to different membrane properties. Second, the role of membrane oxidation in cell membrane vulnerability to the phospholipase (specifically human group IIa) was tested. The temporal onset of lipid peroxidation was assayed during apoptosis. This correlated with the onset of susceptibility to the IIa isoform. Direct oxidizers were then used to verify this result in isolation from other apoptotic membrane changes. Third, biophysical alterations during thapsigargin-induced apoptosis were examined using TMA-DPH and Patman. Data from these probes in artificial bilayers undergoing phase transitions were used to quantify the decrease in interlipid interactions and predict a 50 -- 100-fold increase in the probability of phospholipid protrusions. Patman equilibration kinetics also revealed more molecular detail about the biophysical changes related to susceptibility. Finally, temperature- and ionomyin-induced alterations in membrane properties were compared. Both increased fluidity, but only ionomycin caused susceptibility. Patman equilibration kinetic analysis could distinguish responsible membrane properties. Actin fragmentation during apoptosis or calcium loading is proposed as the mechanism.

No description available.

Urinvägsinfektioner (UVI) är ett vanligt hälsoproblem som drabbar miljontals människor. Den allvarligaste formen, akut pyelonefrit (APN) kan ge svåra komplikationer. Urinvägspatogena Escherichia coli (UPEC) som orsakar APN uttrycker P fimbrier som specifikt binder till glykosphingolipider på ytan av uroepitelceller. Det sätter igång en toll-like receptor 4 (TLR4) beroende men LPS-oberoende immunreaktion. Den roll som interferon beta (IFN-β) spelar vid bakterieinfektioner är inte helt klarlagd men studier som gjorts på IFN-β knockoutmöss visade på en ökad infektionsbenägenhet och svåra njursymptom vid infektion med UPEC. IFN-β uttrycket i uroepitelceller som svar på bakterieinfektion undersöktes. För att ta reda på om uttrycket är P fimbrieberoende infekterades humana A498 njurcarcinomceller med den P fimbrieförsedda pyelonefritstammen CFT073 eller den icke-virulenta asymtomatisk bakterieuristammen E. coli 83972 och inkuberades i 1,5 respektive 4 timmar. Som kontroll användes celler som enbart behandlats med PBS. Uttrycket av IFN-β analyserades med immunofluorescens (IF) och konfokalmikroskopi, samt med Western blot. Resultaten från konfokalmikroskopi visade att celler som exponerats för CFT073 under 4 timmar uttryckte mest IFN-β medan cellerna som utsatts för E. coli 83972 visade på ett omvänt förhållande. Western blot visade på högst uttryck i de E. coli 83972-behandlade cellerna. IFN-β uttrycktes i alla celler, inklusive kontrollcellerna, i någon utsträckning. Det kan betyda att IFN-β även induceras på någon alternativ väg och/eller att det uttrycks konstitutivt av njurepitelceller. / Urinary tract infections (UTI) are a common health concern and affect millions of people. The most severe form of UTI, acute pyelonephritis (APN) is associated with serious complications. Uropathogenic Escherichia coli (UPEC) that cause APN express P fimbriae which specifically bind to glycosphingolipid molecules on the surface of urothelial cells. This triggers a toll-like receptor 4 (TLR4) mediated but LPS-independent innate immune response. The role of interferon beta (IFN-β) in bacterial infections is not well known but experiments with IFN-β knockout mice have shown an increased susceptibility and severe kidney pathology when infected with UPEC. IFN-β induction in urothelial cells in response to bacterial infection was investigated. To find out whether this response is P fimbriae dependent, A498 human kidney carcinoma epithelial cells were exposed to the P fimbriated CFT073 pyelonephritis strain or the non-virulent E. coli 83972 asymptomatic bacteriuria strain and incubated for 1.5 and 4 hours. For control, cells were treated with PBS alone. The IFN-β expression was analysed using immunofluorescence (IF) and confocal microscopy, and Western blot. Confocal microscopy results showed that the response to bacteria was both time- and dose-dependent. The highest IFN-β expression was detected in cells exposed to CFT073 for 4 hours, while cells exposed to E. coli 83972 showed an inverse relationship. Western blot analysis revealed that the highest expression was in the E. coli 83972 stimulated cells. IFN-β was expressed in all cells to some degree, including control cells. This could imply that IFN-β is induced by some other means and/or is constitutively expressed by kidney epithelial cells.