Many of today’s methods for the detection of biomolecules suffer from a high limit ofdetection due to poor signal generation upon recognition of target. By applying andoptimizing proximity ligation assay (PLA) in Western blotting (WB), the limit of detectionhas been lowered down to the picomolar range. In this report I have optimized the differentparameters that affect the signal generation and explored possibilities to increase the ease ofuse, by merging protocol steps and performing signal generating reactions at roomtemperature.

abstract: Measurements of different molecular species from single cells have the potential to reveal cell-to-cell variations, which are precluded by population-based measurements. An increasing percentage of researches have been focused on proteins, for its central roles in biological processes. Immunofluorescence (IF) has been a well-established protein analysis platform. To gain comprehensive insights into cell biology and diagnostic pathology, a crucial direction would be to increase the multiplexity of current single cell protein analysis technologies. An azide-based chemical cleavable linker has been introduced to design and synthesis novel fluorescent probes. These probes allow cyclic immunofluorescence staining which leads to the feasibility of highly multiplexed single cell in situ protein profiling. These highly multiplexed imaging-based platforms have the potential to quantify more than 100 protein targets in cultured cells and more than 50 protein targets in single cells in tissues. This approach has been successfully applied in formalin-fixed paraffin-embedded (FFPE) brain tissues. Multiplexed protein expression level results reveal neuronal heterogeneity in the human hippocampus. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2019

This thesis describes the study of the thermal response of the pyroelectric material named lithium tantalate or LT (LiTaO3) in aid of this material's possible use for temperature measurement. The temperature range studied was between 5-99oC. The sensor was excited using a silicon rubber heater. The lithium tantalate sensor and the rubber heater were enclosed such that the temperature would reach steady state faster. The enclosure was a small insulated box in order to reduce any extraneous effects on the sensor. The output signal of the lithium tantalate sensor was then amplified by using four different amplifying circuits and the voltage output was studied. The amplifying circuits included Current Mode, double Current Mode, Voltage Mode, and a modified Wien Oscillator. Results demonstrated linear dependencies of the voltage output as a function of temperature for the Voltage Mode and the modified Wien Oscillator. Using the modified Wien Oscillator amplifying circuit the slope of the line a 2.1mV/oC and for the Voltage Mode the slope was 1mV/oC. For both cases it was found that the range for the standard deviation of the measurements was 0.5< The data showed that the lithium tantalate sensor could be used as a temperature measuring device for the range mentioned above. The resolution of the data is high enough to be able to be detected with modern measuring devices and the standard deviation is low enough to allow for such measurements. Moreover, the linear dependence of the data allows for accurate measurements at each temperature within the range.

No description available.

京都大学 / 新制・論文博士 / 博士(人間健康科学) / 乙第13562号 / 論人健博第12号 / 新制||人健||8(附属図書館) / (主査)教授 高桑 徹也, 教授 藤井 康友, 教授 長尾 美紀 / 学位規則第4条第2項該当 / Doctor of Human Health Sciences / Kyoto University / DFAM

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.

Immunoassays are analytical methods that use the highly specific binding of antibodies in order to detect and quantify an analyte. The technique has become a staple in modern biopharmaceutical research and diagnostics, however the measurement of biomarkers like dysregulated cytokines require ultra-sensitive immunoassays that can detect molecules at sub pg/mL concentrations. One such method is the Exazym® signal amplification. Based on a method called Binding Oligo Ladder Detection (BOLD), it is a set of add-on reagents where a primer is conjugated to a detection antibody which is then combined with a template, polymerase and modified DNA nucleotides to generate a oligonucleotide ladder that is detected with a secondary detection antibody; this amplifies the signal by a factor of 10-100 in an existing immunoassay.  By applying this method to the Gyrolab® microfluidic immunoassay system, a sensitivity increase of 880x-1800x was achieved between a pre-synthesised BOLD product and the polymerised BOLD product. Several key factors for successful polymerisation in the microfluidic system were identified: adding the template separately before the polymerase and using a buffer with low ionic strength for the secondary detection antibody. Applying the BOLD amplification to an existing Gyrolab TNF-α assay only resulted in similar sensitivity as previous methods however. This report demonstrates that BOLD amplification can be successfully performed in a flow-through format on miniaturized affinity columns in the Gyrolab system to increase the sensitivity by orders of magnitude, where both the immunoassay and the amplification steps are automated in the system. However, further optimisation is needed for application in biomarker assays.

Les changements évolutifs nous instruisent sur les nombreuses innovations permettant à chaque organisme de maximiser ses aptitudes en choisissant le partenaire approprié, telles que les caractéristiques sexuelles secondaires, les patrons comportementaux, les attractifs chimiques et les mécanismes sensoriels y répondant. L'haploïde de la levure Saccharomyces cerevisiae distingue son partenaire en interprétant le gradient de la concentration d'une phéromone sécrétée par les partenaires potentiels grâce à un réseau de protéines signalétiques de type kinase activées par la mitose (MAPK). La décision de la liaison sexuelle chez la levure est un événement en "tout–ourien", à la manière d'un interrupteur. Les cellules haploïdes choisissent leur partenaire sexuel en fonction de la concentration de phéromones qu’il produit. Seul le partenaire à proximité sécrétant des concentrations de phéromones égales ou supérieures à une concentration critique est retenu. Les faibles signaux de phéromones sont attribués à des partenaires pouvant mener à des accouplements infructueux. Notre compréhension du mécanisme moléculaire contrôlant cet interrupteur de la décision d'accouplement reste encore mince. Dans le cadre de la présente thèse, je démontre que le mécanisme de décision de la liaison sexuelle provient de la compétition pour le contrôle de l'état de phosphorylation de quatre sites sur la protéine d'échafaudage Ste5, entre la MAPK, Fus3, et la phosphatase,Ptc1. Cette compétition résulte en la dissociation de type « intérupteur » entre Fus3 et Ste5, nécessaire à la prise de décision d'accouplement en "tout-ou-rien". Ainsi, la décision de la liaison sexuelle s'effectue à une étape précoce de la voie de réponse aux phéromones et se produit rapidement, peut-être dans le but de prévenir la perte d’un partenaire potentiel. Nous argumentons que l'architecture du circuit Fus3-Ste5-Ptc1 génère un mécanisme inédit d'ultrasensibilité, ressemblant à "l'ultrasensibilité d'ordre zéro", qui résiste aux variations de concentration de ces protéines. Cette robustesse assure que l'accouplement puisse se produire en dépit de la stochasticité cellulaire ou de variations génétiques entre individus.Je démontre, par la suite, qu'un évènement précoce en réponse aux signaux extracellulaires recrutant Ste5 à la membrane plasmique est également ultrasensible à l'augmentation de la concentration de phéromones et que cette ultrasensibilité est engendrée par la déphosphorylation de huit phosphosites en N-terminal sur Ste5 par la phosphatase Ptc1 lorsqu'elle est associée à Ste5 via la protéine polarisante, Bem1. L'interférence dans ce mécanisme provoque une perte de l'ultrasensibilité et réduit, du même coup, l'amplitude et la fidélité de la voie de réponse aux phéromones à la stimulation. Ces changements se reflètent en une réduction de la fidélité et de la précision de la morphologie attribuable à la réponse d'accouplement. La polarisation dans l'assemblage du complexe protéique à la surface de la membrane plasmique est un thème général persistant dans tous les organismes, de la bactérie à l'humain. Un tel complexe est en mesure d'accroître l'efficacité, la fidélité et la spécificité de la transmission du signal. L'ensemble de nos découvertes démontre que l'ultrasensibilité, la précision et la robustesse de la réponse aux phéromones découlent de la régulation de la phosphorylation stoichiométrique de deux groupes de phosphosites sur Ste5, par la phosphatase Ptc1, un groupe effectuant le recrutement ultrasensible de Ste5 à la membrane et un autre incitant la dissociation et l'activation ultrasensible de la MAPK terminal Fus3. Le rôle modulateur de Ste5 dans la décision de la destinée cellulaire étend le répertoire fonctionnel des protéines d'échafaudage bien au-delà de l'accessoire dans la spécificité et l'efficacité des traitements de l'information. La régulation de la dynamique des caractères signal-réponse à travers une telle régulation modulaire des groupes de phosphosites sur des protéines d'échafaudage combinées à l'assemblage à la membrane peut être un moyen général par lequel la polarisation du destin cellulaire est obtenue. Des mécanismes similaires peuvent contrôler les décisions cellulaires dans les organismes complexes et peuvent être compromis dans des dérèglements cellulaires, tel que le cancer. Finalement, sur un thème relié, je présente la découverte d'un nouveau mécanisme où le seuil de la concentration de phéromones est contrôlé par une voie sensorielle de nutriments, ajustant, de cette manière, le point prédéterminé dans lequel la quantité et la qualité des nutriments accessibles dans l'environnement déterminent le seuil à partir duquel la levure s'accouple. La sous-unité régulatrice de la kinase à protéine A (PKA),Bcy1, une composante clé du réseau signalétique du senseur aux nutriments, interagit directement avec la sous-unité α des petites protéines G, Gpa1, le premier effecteur dans le réseau de réponse aux phéromones. L'interaction Bcy1-Gpa1 est accrue lorsque la cellule croit en présence d'un sucre idéal, le glucose, diminuant la concentration seuil auquel la décision d'accouplement est activée. Compromettre l'interaction Bcy1-Gpa1 ou inactiver Bcy1 accroît la concentration seuil nécessaire à une réponse aux phéromones. Nous argumentons qu'en ajustant leur sensibilité, les levures peuvent intégrer le stimulus provenant des phéromones au niveau du glucose extracellulaire, priorisant la décision de survie dans un milieu pauvre ou continuer leur cycle sexuel en choisissant un accouplement. / Evolution has resulted in numerous innovations that allow organisms to maximize their fitness by choosing particular mating partners, including secondary sexual characteristics, behavioural patterns, chemical attractants and corresponding sensory mechanisms. The haploid yeast Saccharomyces cerevisiae selects mating partners by interpreting the concentration gradient of pheromone secreted by potential mates through a network of mitogen-activated protein kinase (MAPK) signaling proteins. The mating decision in yeast is an all-or-none, or switch-like, response that allows cells to make accurate decisions about which among potential partners to mate with and to filter weak pheromone signals, thus avoiding inappropriate commitment to mating by responding only at or above critical concentrations when a mate is sufficiently close. The molecular mechanisms that govern the switch-like mating decision are poorly understood. In this thesis I demonstrate that the switching mechanism arises from competition between the MAPK Fus3 and a phosphatase Ptc1 for control of the phosphorylation state of four sites on the scaffold protein Ste5. This competition results in a switch-like dissociation of Fus3 from Ste5 that is necessary to generate the switch-like mating response. Thus, the decision to mate is made at an early stage in the pheromone pathway and occurs rapidly, perhaps to prevent the loss of the potential mate to competitors. We argue that the architecture of the Fus3–Ste5–Ptc1 circuit generates a novel ultrasensitivity mechanism that resembles “zero-order ultrasensitivity”, which is robust to variations in the concentrations of these proteins. This robustness helps assure that mating can occur despite stochastic or genetic variation between individuals. I then demonstrate that during the mating response, an early event of Ste5 recruitment to plasma membrane is ultrasensitive and that it is generated by dephosphorylation of eight N-terminal phosphosites on Ste5 by the phosphatase Ptc1 when associated with Ste5 via the polarization protein Bem1. Interference with this mechanism results in loss of ultrasensitivity and reduced amplitude and therefore fidelity of the pheromone signaling response. These changes are reflected in reduced fidelity and accuracy of the morphogenic mating response. Polarized assembly of signaling protein complexes at the plasma membrane surface is a general theme recapitulated in all organisms from bacteria to humans. Such complexes can increase the efficiency, fidelity and specificity of signal transduction. Together with our previous findings, our results demonstrate that ultrasensitivity, accuracy and robustness of the pheromone response occurs through regulation of the stoichiometry of phosphorylation of two clusters of phosphosites on Ste5, by Ptc1, one cluster mediating ultrasensitive recruitment of Ste5 to the membrane and the other, ultrasensitive dissociation and activation of the terminal MAP kinase Fus3. The role of Ste5 as a direct modulator of a cell-fate decision expands the functional repertoire of scaffold proteins beyond providing specificity and efficiency of information processing. Regulation of dynamic signal-response characteristics through such modular regulation of clusters of phosphosites may be a general means by which cell fate decisions are achieved. Similar mechanisms may govern cellular decisions in higher organisms and be disrupted in cancer. Finally, in a related theme, I present the discovery of a novel mechanisms by which the threshold of pheromone response is controlled by a nutrient-sensing pathway, thus adjusting the set-point at which the quantity and quality of nutrients available in the environment set the threshold of pheromone at which yeast will mate. The regulatory subunit of protein kinase A (PKA), Bcy1, a key component of a nutrient sensing signaling network, directly interacts with the α subunit of G-protein, Gpa1, the primary effector of the pheromone signaling network. The Bcy1-Gpa1 interaction is enhanced when cells are grown in their ideal carbon source glucose, lowering the threshold concentration at which the mating response is activated. Disruption of Bcy1-Gpa1 interaction or Bcy1 deletion increased the threshold concentration for the mating response. We argue that by adjusting their sensitivity, yeast can integrate pheromone stimulus with glucose levels and prioritize decisions to survive in a nutrient-starved environment or to continue their sexual cycle by mating.

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.

No description available.