The G protein-coupled receptor (GPCR) family is composed of hundreds of members and is expressed in eukaryotes. Each GPCR has seven transmembrane domains and is in charge of sensing changes from the environment, transducing signals, and activating a series of biological responses. The signal transduction pathway of the receptor starts from sensing outside signal and then activates G proteins. This signaling requires a tight control for activation without which impaired cellular function leads to pathology. We have used the pheromone alpha-factor receptor (Ste2p) of the yeast Saccharomyces cerevisiae as a model system to understand ligand binding, receptor activation, and G protein interaction. One method we have used to study ligand binding is to incorporate the photo-reactive crosslinker p-benzoyl-L-phenylalanine (Bpa) into Ste2p to capture alpha-factor. This powerful tool requires the incorporation of Bpa, an unnatural amino acid, into Ste2p by a special genetic manipulation designed in the lab of Peter Schulz (Scripps Institute) and adapted by our lab for Ste2p. Another method to study ligand binding that we have adapted for use in our system is to incorporate a chemical crosslinker [3,4-dihydroxylphenylacetyl (DHPA)] into alpha-factor for periodate-mediated crosslinking to Ste2p. The interacting domain between alpha-factor and transmembrane domain 2 to 3 of Ste2p was identified after DOPAC crosslinking, cyanogen bromide digestion and MALDI-TOF mass spectrometry. After ligand binding, signal transduction is mediated by the interaction of activated Ste2p with its G protein (Gpa1p). We studied this interaction by replacing natural residues in the intracellular loop 3 of Ste2p and C-terminal end of Gpa1p with cysteine and then determining disulfide crosslinking between Ste2p and Gpa1p. Some residues were found to be in close proximity and displayed different interacting patterns due to conformational changes of the receptor upon ligand binding. The information we gathered here allows us to understand more about the physical interactions of alpha-factor, Ste2p, and Gpa1p and provides us insights about the initiation and activation of the signal transduction pathway of a peptide ligand receptor.

Compared to single-mode VCSELs, multimode VCSELs have not received much attention in models and characterizations for functional parameters, despite making up the majority of commercially available VCSELs [1]. In particular, the extraction of the linewidth enhancement factor for multimode VCSELs has been overlooked, likely due to difficulties in measurement. Additionally, multimode models for VCSELs have, until recently, omitted spectral characteristics such as linewidth [2]. This is the first work to report a measured linewidth enhancement factor value (lower bound) for a multimode VCSEL. A characterization for the functional parameters of a red multimode vertical-cavity surface-emitting laser (VCSEL) is shown herein. The extracted values form a complete working set of parameters for the laser rate equations. The techniques employed for extracting values include frequency responses, power versus current fittings, and optical spectral measurements. From the frequency responses at various bias currents, the relaxation oscillation frequency and damping factor are found. The power versus current curve is fitted to find parameters including the modal spontaneous emission rate and carrier density at threshold. The spectral measurements are used for evaluating the linewidth enhancement factor (LEF) also known as the alpha factor or Henry factor. These 5 methods have been applied previously to characterizing single-mode VCSELs [3]–[5]. The experimentally extracted parameters herein are important for creating accurate models and simulations for multimode VCSELs. Improved multimode VCSEL models are necessary for improving optical communication, especially for short-range optical interconnects [2]. The measured parameters for the characterized VCSEL are comparable to similar single-mode VCSELs characterized in other works. This is promising because multi-mode VCSELs have higher output power than their single-mode counterparts, thus these results may aid in improving short-range optical interconnects.

Les performances des bassins de stations d'épuration sont directement liées au transfert d'oxygène de bulles d'air généralement insufflées par des aérateurs vers des micro-organismes capables de détériorer certains polluants des eaux usées. Une prédiction précise du transfert de matière de l'oxygène dans ces bassins reste toutefois encore délicate. L'objectif de cette thèse est de développer des outils d'analyse aux interfaces afin de mieux appréhender localement les différents mécanismes régissant le transfert de matière gaz/liquide. Cette simplification se base sur l'étude du transfert d'oxygène : (i) pour une bulle millimétrique isolée, (ii) dans un liquide au repos, (iii) sans particules solides (système diphasique). À l'aide de techniques de visualisation, il est possible d'évaluer l'influence de la composition de la phase liquide sur le coefficient de transfert côté́ liquide par PLIF (Fluorescence Induite par un Plan Laser) et sur le comportement hydrodynamique de la bulle (diamètre, vitesse, forme) par caméra rapide. L'influence du diamètre de la bulle sur le transfert de matière visualisé est également évaluée. Le coefficient de transfert côté liquide n'étant pas le seul paramètre représentatif du transfert de matière, des techniques expérimentales spécifiques sont développées afin d'estimer avec précision le coefficient de diffusion de l'oxygène dans la phase liquide d'intérêt ainsi que sa concentration à saturation au travers d'une interface gaz/liquide plane en cellule de Hele-Shaw. Via ces différentes techniques expérimentales développées durant la thèse, une cartographie précise de l'influence de la phase liquide sur le transfert d'oxygène est établie en évaluant dans un premier temps l'influence de certains composants isolés (sel, glucose, alcool, tensio-actifs, médicaments, etc.). Ainsi, il sera observé que certains composants, comme les tensio-actifs, peuvent fortement diminuer le transfert de matière. Les corrélations généralement utilisées afin de caractériser ce transfert de matière ne parvenant pas toujours à représenter de telles diminutions, ces résultats seront par la suite comparés à ceux obtenus dans des eaux de stations d'épuration afin d'approfondir la compréhension des différents mécanismes limitant ce transfert d'oxygène / The performance of tanks in sewage treatment plants is highly related to the oxygen transfer from air bubbles, usually injected through di��users, to microorganisms able to degrade pollutants contained in wastewaters. However, characterizing accurately the oxygen mass transfer in such processes is still a challenging issue mainly because of the liquid phase complexity. The aim of this PhD manuscript is to develop specific techniques to better understand the various mechanisms that locally govern the gas/liquid mass transfer process. This study focuses on the oxygen mass transfer, (i) from a single bubble, (ii) rising in a stagnant liquid, (iii) free from solid particles (biphasic system). Efficient visualization techniques allow the impact of the liquid phase composition on the liquid- side mass transfer coefficient (using PLIF (Planar Laser-Induced Fluorescence)) and on the bubble hydrodynamic behavior (bubble diameter, shape, velocity) using high-speed cameras to be evaluated. Moreover, the impact of the bubble diameter on the mass transfer visualized is considered. Since the liquid-side mass transfer coefficient is often not sufficient to fully characterize mass transfer, specific experimental techniques, based on planar gas/liquid interfaces in Hele-Shaw cells, are proposed to estimate the diffusion coefficient of oxygen and the oxygen saturation concentration in several liquids of interest. Using the visualization techniques developed during this PhD thesis, the impact of the liquid phase on the oxygen mass transfer is evaluated by introducing different compounds in the liquid phase (salts, glucose, alcohol, surfactants, drugs, etc.). It is interesting to note that some compounds, such as surfactants, can drastically bother the oxygen mass transfer. Since the correlations classically used to characterize mass transfer cannot explain such performance degradation, the results obtained (with liquids whose composition is known) are finally compared to those obtained with wastewater from sewage treatment plants to better understand the factors that can alter the oxygen mass transfer

Heterochromatin in the budding yeast Saccharomyces cerevisiae is composed of polymers of the SIR (Silent Information Regulator) complex bound to nucleosomal DNA. Assembly of heterochromatin requires all three proteins of the Sir complex: the histone deacetylase Sir2, and histone binding proteins Sir3 and Sir4. Heterochromatin establishment requires passage through at least one cell cycle, but is not dependent on replication. Inhibition of chromatin modifying enzymes may be a mechanism for how cells limit assembly. Dot1 dependent methylation of H3K79 is suggested to inhibit de novo assembly. Halving the levels of Sir4 in cells causes a loss of silencing, suggesting that Sir4 protein abundance regulates the assembly of heterochromatin. We examine de novo assembly using a single cell assay. Half the level of Sir4 affects establishment, but not the maintenance, of silencing at HM loci. Additional Sir4 accelerates the rate of assembly. Epistasis analysis suggests that Dot1 dependent chromatin modification may act upstream of Sir4 abundance. We hypothesize that dot1Δ mutants speed assembly by disrupting telomeric heterochromatin, which liberates Sir4 to act at the HM loci. Deletion of YKU70, which specifically disrupts telomeric silencing, also speeds de novo assembly, without altering the methylation of histone H3. Consistent with our model, we have shown that Sir4 abundance falls during pheromone and stationary phase arrests after which several cell cycles are required before silencing can be reestablished.

This work was performed in the framework of two application-oriented research projects that focus on the generation and evaluation of fluorescent Saccharomyces (S.) cerevisiae-based sensor and reporter cells for white biotechnology as well as the extension of the conventional single-cell/single-construct principle of ordinary yeast biosensor approaches. Numerous products are currently generated by biotechnological processes which require continuous and precise process control and monitoring. These demands are only partially met by physical or physiochemical sensors since they measure parameters off-line or use surrogate parameters that consequently provide only indirect information about the actual process performance. Biosensors, in particular whole cell-based biosensors, have the unique potential to near-line and long-term monitor parameters such as nutrient availability during fermentation processes. Moreover, they allow for the assessment of an analyte’s biological relevance. Prototype yeast sensor and reporter strains derived from common laboratory strains were transformed with multicopy expression plasmids that mediate constitutive or inducible expression of a fluorescence reporter gene. Performance of these cells was examined by various qualitative and quantitative detection methods – representative of putative transducer technologies. Analyses were performed on the population level by microplate reader-based fluorometry and Western blot as well as on the single-cell level by fluorescence microscopy and flow cytometry. ‘Signature’ promoters that are activated or repressed during particular nutrient-limited growth conditions were selected in order to generate yeast nutrient sensor strains for monitoring the biological availability of nitrogen, phosphorus or sulphur. For each category, at least one promoter mediating at least threefold changed green fluorescence levels between sensor cells in non-limited and nutrient-limited conditions was identified. Sensor strains were evaluated in detail regarding sensitivity, analyte selectivity and the ability to restore basic fluorescence after shift from nutrient-limited to non-limited conditions (regeneration). The applicability for bioprocess monitoring purposes was tested by growth of yeast nutrient sensor cells in microalgae media and supernatants. Despite successful proof of principle, numerous challenges still need to be solved to realise prospective implementation in this field of white biotechnology. The major drawback of plasmid-borne detection constructs is a high fluorescence variance between individual cells. By generation of a nitrogen sensor strain with a genome-integrated detection construct, uniform expression on the single-cell level and simultaneous maintenance of basic properties (ability of fluorescence induction/regeneration and lack of cross-reactivity) was achieved. However, due to the singular detection construct per cell, significantly weaker overall fluorescence was observed. The traditional single-cell/single-construct approach was expanded upon in two ways. Firstly, a practical dual-colour sensor strain was created by simultaneous, constitutive expression of a red fluorescence reporter gene in green fluorescent nitrogen sensor cells. Secondly, an innovative cellular communication and signal amplification system inspired by the natural S. cerevisiae pheromone system and mating response was established successfully. It features the yeast pheromone alpha-factor as a trigger and alpha-factor-responsive reporter cells which express a fluorescence reporter gene from the pheromone-inducible FIG1 promoter as an output signal. The system was functional both with synthetic and cell-secreted alpha-factor, provided that recombinant cells were deleted for the alpha-factor protease Bar1p. Integration of amplifier cells which secrete alpha-factor in response to stimulation with the pheromone itself could increase the system\'s sensitivity further. Signal amplification was demonstrated for phosphorus sensor cells as a proof of concept. Therefore, the alpha-factor-based cellular communication and signal amplification system might be useful in applications that suffer from poor signal yield. Due to its modular design, the system could be applied in basically any cell-based biosensor or sensor-actor system. Immobilisation of the generated sensor and reporter cells in transparent natural polymers can be beneficial considering biosensor fabrication. Functionality of sensor and reporter cells in calcium-alginate beads or nano-printed arrays was successfully demonstrated. For the latter setup, fluorescence scanning and software-assisted fluorescence quantification was applied as a new detection method. In an experiment using an agarose-based two-compartment setup proposed by Jahn, 2011, properties of the alpha-factor-based cellular communication and signal amplification system after immobilisation were tested. These studies provide an initial experimental basis for an appropriate geometry of miniaturised immobilisation matrices with fluorescent yeast sensor and reporter cells in prospective biosensor designs.

This work was performed in the framework of two application-oriented research projects that focus on the generation and evaluation of fluorescent Saccharomyces (S.) cerevisiae-based sensor and reporter cells for white biotechnology as well as the extension of the conventional single-cell/single-construct principle of ordinary yeast biosensor approaches. Numerous products are currently generated by biotechnological processes which require continuous and precise process control and monitoring. These demands are only partially met by physical or physiochemical sensors since they measure parameters off-line or use surrogate parameters that consequently provide only indirect information about the actual process performance. Biosensors, in particular whole cell-based biosensors, have the unique potential to near-line and long-term monitor parameters such as nutrient availability during fermentation processes. Moreover, they allow for the assessment of an analyte’s biological relevance. Prototype yeast sensor and reporter strains derived from common laboratory strains were transformed with multicopy expression plasmids that mediate constitutive or inducible expression of a fluorescence reporter gene. Performance of these cells was examined by various qualitative and quantitative detection methods – representative of putative transducer technologies. Analyses were performed on the population level by microplate reader-based fluorometry and Western blot as well as on the single-cell level by fluorescence microscopy and flow cytometry. ‘Signature’ promoters that are activated or repressed during particular nutrient-limited growth conditions were selected in order to generate yeast nutrient sensor strains for monitoring the biological availability of nitrogen, phosphorus or sulphur. For each category, at least one promoter mediating at least threefold changed green fluorescence levels between sensor cells in non-limited and nutrient-limited conditions was identified. Sensor strains were evaluated in detail regarding sensitivity, analyte selectivity and the ability to restore basic fluorescence after shift from nutrient-limited to non-limited conditions (regeneration). The applicability for bioprocess monitoring purposes was tested by growth of yeast nutrient sensor cells in microalgae media and supernatants. Despite successful proof of principle, numerous challenges still need to be solved to realise prospective implementation in this field of white biotechnology. The major drawback of plasmid-borne detection constructs is a high fluorescence variance between individual cells. By generation of a nitrogen sensor strain with a genome-integrated detection construct, uniform expression on the single-cell level and simultaneous maintenance of basic properties (ability of fluorescence induction/regeneration and lack of cross-reactivity) was achieved. However, due to the singular detection construct per cell, significantly weaker overall fluorescence was observed. The traditional single-cell/single-construct approach was expanded upon in two ways. Firstly, a practical dual-colour sensor strain was created by simultaneous, constitutive expression of a red fluorescence reporter gene in green fluorescent nitrogen sensor cells. Secondly, an innovative cellular communication and signal amplification system inspired by the natural S. cerevisiae pheromone system and mating response was established successfully. It features the yeast pheromone alpha-factor as a trigger and alpha-factor-responsive reporter cells which express a fluorescence reporter gene from the pheromone-inducible FIG1 promoter as an output signal. The system was functional both with synthetic and cell-secreted alpha-factor, provided that recombinant cells were deleted for the alpha-factor protease Bar1p. Integration of amplifier cells which secrete alpha-factor in response to stimulation with the pheromone itself could increase the system\'s sensitivity further. Signal amplification was demonstrated for phosphorus sensor cells as a proof of concept. Therefore, the alpha-factor-based cellular communication and signal amplification system might be useful in applications that suffer from poor signal yield. Due to its modular design, the system could be applied in basically any cell-based biosensor or sensor-actor system. Immobilisation of the generated sensor and reporter cells in transparent natural polymers can be beneficial considering biosensor fabrication. Functionality of sensor and reporter cells in calcium-alginate beads or nano-printed arrays was successfully demonstrated. For the latter setup, fluorescence scanning and software-assisted fluorescence quantification was applied as a new detection method. In an experiment using an agarose-based two-compartment setup proposed by Jahn, 2011, properties of the alpha-factor-based cellular communication and signal amplification system after immobilisation were tested. These studies provide an initial experimental basis for an appropriate geometry of miniaturised immobilisation matrices with fluorescent yeast sensor and reporter cells in prospective biosensor designs.

Le futur système de transmission multicanaux (WDM) pourrait mettre à profit l'utilisation d'amplificateurs optiques à semi-conducteurs (SOA), pour bénéficier notamment de leur grande bande passante optique pour l'amplification du signal. Dans ce travail, nous étudions l’influence des SOA sur la transmission cohérente de signaux OFDM optiques (CO OFDM). Cette technique, récemment proposée, permet à la fois d’augmenter l'efficacité spectrale de la transmission et de compenser les imperfections linéaires du canal optique. Nous avons développé, dans ce travail, une chaîne expérimentale de transmission de signaux à formats de modulation complexes tout-optiques et une plateforme de simulation au niveau système. Les résultats obtenus par simulation, au niveau composant et au niveau système, sont en très bon accord avec ceux obtenus par les mesures expérimentales à la fois pour des formats de modulation QPSK et QPSK CO-OFDM. À travers différentes mesures et simulations, l’étude a permis de cerner clairement l’influence des paramètres du SOA sur la qualité de transmission des données. Les non-linéarités induites par le SOA, telles que le couplage phase amplitude, l’auto modulation du gain et de la phase (SGM et SPM), la modulation croisée du gain et de la phase (XGM et XPM) et le mélange à quatre ondes (FWM), jouent de façon importante sur les performances de ce format de modulation multi-porteuses ; leur influence a donc été analysée avec précision. Les connaissances acquises permettront à l’avenir d’une part de mieux définir les conditions d’utilisation des SOA dans les réseaux de transmission et d’autre part aideront à l’optimisation de nouvelles structures de SOA conçues pour la transmission de données à très haut débit utilisant des formats de modulation complexes. / Future wavelength division multiplexing (WDM) systems might take advantage from the use of semiconductor optical amplifiers (SOA), especially to benefit from their large optical bandwidth for signal amplification. In this work, we study the influence of SOAs on the coherent optical-OFDM (CO OFDM) transmission system. This recently proposed technique allows both to increase the spectral efficiency of the transmission and to compensate the linear imperfections of the optical channel. In this work, we have developed an experimental setup for signal transmission operating with advanced optical modulation formats and a system level simulation platform. Simulation results, both at the component level and at the system level, are in very good agreement with those obtained from experimental measurements in the case of both QPSK and QPSK CO-OFDM signals. The study has clearly identified, through various measurements and simulations, the influence of SOA parameters on the quality of data transmission. Nonlinearities induced by the SOA, such as phase-amplitude coupling, self gain and phase modulation (SGM and SPM), cross gain and phase modulation (XGM and XPM) and the four-wave mixing (FWM) affect the performances of this multicarrier modulation format. Their influence is studied very precisely in this work. This acquired knowledge will allow, on the one hand, better defining the conditions of use of SOAs in the transmission networks and, on the other hand, helping to optimize new structures of SOA designed for very high bit rate data transmissions using complex modulation formats.