Dissertations / Theses on the topic 'Yeast fluorescence'

The advent and establishment of systems biology has cemented the idea that real understanding of biological systems requires quantitative models, that can be integrated to provide a complete description of the cell and its complexities. At the same time, synthetic biology attempts to leverage such quantitative models to efficiently engineer novel, predictable behaviour in biological systems. Together, these advances indicate that the future understanding and application of biology will require the ability to create, parameterise and discriminate between quantitative models of cellular processes in a rigorous and statistically sound manner. In this thesis we take the regulation of GAL1 expression in Saccharomyces cerevisiae as a test case and look at all aspects of this process: from reporter selection to data acquisition and statistical analysis. In chapter B we will discuss optimal fluorescent reporter selection and construction for investigating transcriptional dynamics, as well as procedures for quantifying and correcting the various sources of error in our microscope system. In chapter 3 we will describe software developed to analyse fluorescent microscopy images and convert them to gene expression data. A number of iterations of the software are tested against manually curated data sets, and the measurement error produced by its imperfections is quantified and discussed. In chapter 4 a method, based on fluctuations in photobleaching, is developed for quantifying both measurement error and the relationship between protein concentration and measured fluorescence. The method is refined and its efficacy discussed. In the last section I make a preliminary application of these methods to investigating the regulatory effect of the GAL10-lncRNA. Interesting phenomena are observed and further investigated using two new strains: genetic variants expressing a fluorescent reporter from the GAL1 promoter, one harbouring a wild type GAL1 promoter and one in which the binding site for the Gal10 noncoding RNA has been removed. The methods developed throughout the thesis are applied and the data analysed. A heterogeneous response, distinguishable between the two strains, is observed and related to cell-to-cell variations in growth rate.

Within the dissertation thesis “Study of Biological Material Attributes by Using Image Analysis Methods”, attention is focused on monitoring of the application of image analysis methods, mostly a fractal analysis, in studying the properties of various yeast species. The thesis includes determining the number of yeast cells and vegetative propagation of yeast using fractal parameters – fractal measure D and fractal dimension K. Attention is also paid not only to the application of the existing image analysis methods, but also to their renovation. The obtained images were evaluated using the box counting method specified by implementation of wavelet transformation. To monitor yeast cells for a longer time, it was first necessary to prepare a suitable microscopic preparation. To distinguish live and dead cells, the following fluorescent dyes were used: acridine orange, fluorescein diacetate, FUN-1, and Calcofluor White M2R. The images of yeast cells were recorded using a still camera or a CCD camera and microscope. Clips of the same size were obtained from the acquired digital photographs and processed by the HarFA program developed at the Faculty of Chemistry, Brno University of Technology. On the results it is possible to see a change in the fractal dimension depending on time, i.e. on the change of a budding cell structure, or to determine the number and radius of yeast cells upon predefined conditions.

Biology in the past two decades has shifted away from qualitative observation towards quantitative data, and away from reductionism towards viewing the phenomena under study as systems. Developments in fluorescent reporters, experimental instrumentation, and computational power have been integral to this change. A key feature of this transformation has been a substantial increase in the use of mathematical models to explore and verify our understanding of complicated biological mechanisms. This thesis looks at three different aspects of the systems modelling approach: data acquisition, model creation and evaluation, and measurement of parameter values. The system under study for the first two parts is the GAL pathway of the yeast Saccharomyces cerevisiae, a well-known model for genetic regulation. Despite this pathway being relative simple and well studied, various questions remain about the details of its regulatory mechanism. In the first part, I describe methodology for acquiring dynamic protein expression data. I use the green fluorescent protein (GFP) as a reporter and make measurements from cell populations growing in a microplate reader. Of particular interest is a technique I introduce for removing the autofluorescence that contaminates the GFP fluorescence signal. My technique makes it possible to detect expression even from very weakly expressed proteins. In the second part, I have developed a basic, deterministic model of the GAL pathway, which I then fit to dynamic protein expression data. I use the model to demonstrate that based on available data, the current understanding of the GAL pathway does capture a wide range of GAL system behaviours. I also identify and explore the GAL network's sensitivity to the relative levels of inducer and repressor proteins and discuss future directions for experiments and model development. In the third part, I present theoretical work that addresses the challenge of measuring protein-protein interactions in vivo from Förster resonance energy transfer (FRET) fluorescence data. I designed a computational tool that uses a Bayesian statistical framework to infer the dissociation constant and FRET efficiency from FRET data. One key advantage of this approach is that it produces probability distributions for these parameters of interest, revealing the uncertainty in the estimates obtained. I also demonstrate the experimental conditions, such as variations in levels of FRET donors and acceptors, that are necessary for the inference of the dissociation constant and FRET efficiency to be possible.
Au cours des deux dernières décennies, la biologie est passée d'une science réductionniste fondée sur l'observation qualitative à une science quantitative traitant les phénomènes biologiques comme des systèmes. Le développement de gènes rapporteurs fluorescents, d'instruments expérimentaux sophistiqués et de la puissance de calcul des ordinateurs ont été des éléments garants de cette transformation. Plus particulièrement, un élément clé de cette transformation a été l'utilisation croissante de modèles mathématiques pour explorer et vérifier notre compréhension de mécanismes biologiques complexes. Cette thèse traite de trois aspects de la modélisation des systèmes: l'acquisition de données, la création et l'évaluation d'un modèle et la mesure des valeurs de ses paramètres.Le système étudié dans les deux premières parties de cette thèse est la voie métabolique du galactose (GAL) chez la levure Saccharomyces cerevisiae. Cette voie est un exemple bien connu de régulation génétique. Bien qu'elle soit relativement simple et déjà bien étudiée, plusieurs questions subsistent quant aux détails reliés à son mécanisme de régulation. Dans la première partie, je décris la méthodologie pour l'acquisition de données dynamiques d'expression de protéines. J'utilise la protéine fluorescente verte (GFP) comme rapporteur et effectue des mesures sur des populations de cellules croissant dans un lecteur de microplaques. Notamment, je présente une technique permettant d'éliminer l'autofluorescence qui contamine le signal de fluorescence de la GFP. Cette technique permet même la détection de l'expression de protéines très faiblement exprimées. Dans la deuxième partie, je développe un modèle déterministe de base de la voie GAL, que je raccorde par la suite à des données dynamiques d'expression de protéines. J'utilise ce modèle pour démontrer qu'en se basant sur les données disponibles la compréhension théorique actuelle de la voie GAL capture une grande partie des comportements réels du système. Je discute aussi de possibilités pour l'élaboration subséquente du modèle. Dans la troisième partie, je présente un travail théorique qui traite de la difficulté de mesurer in vivo les interactions entre protéines à partir de données fluorescentes résultant du transfert d'énergie par résonance de type Förster (FRET). Je conçois un outil de calcul qui utilise les statistiques bayésiennes pour déduire la constante de dissociation et l'efficacité du FRET de données FRET. Un avantage clé de cette approche est qu'elle produit des distributions de probabilité pour ces paramètres d'intérêt, révélant l'incertitude des estimations obtenues. Je démontre aussi les conditions expérimentales, telles que les variations dans les concentrations de donneurs et d'accepteurs du FRET, requises pour permettre l'inférence de la constante de dissociation et de l'efficacité du FRET.

The effective and efficient generation of both antibodies and antibody fragments to proteins of interest is vital, as antibodies and antibody fragments are required for an ever-increasing variety of therapeutic, diagnostic and analytical applications. The single chain variable fragment (scFv) is an antibody fragment consisting of a heavy chain variable region (VH) and a light chain variable region (VL) joined together by a flexible polypeptide linker. In 2003, Feldhaus et al. developed a nonimmune human scFv surface display library, in Saccharomyces cerevisiae , containing 109 different scFvs. Cells in the library expressing scFvs of interest can be isolated using magnetic cell sorting (MCS) and fluorescence activated cell sorting (FACS).
The reduced size of the scFv relative to the intact IgG allows it to penetrate tissue with greater ease and therefore reach epitopes within both tissue and cells that would otherwise remain inaccessible. As a result, one possible scFv application is the study of cartilage destruction by proteases that occurs in both normal joint development and arthritis. Antibody fragments would allow for cartilage degradative processes to be studied in vivo . Fluorescently tagged scFvs could penetrate intact cartilage tissue, bind to epitopes and then be localized using techniques such as dual photon confocal microscopy. This would not be possible using IgG molecules.
The yeast library developed by Feldhaus et al. was obtained for the potential isolation of cells expressing scFvs to cartilage neoepitopes. While found to possess an inherent Candida parapsilosis contamination, the surface display library was screened using three peptide-ovalbumin-biotin complexes. Peptides corresponding to observed cartilage neoepitopes were bound to biotinylated ovalbumin and added to the library for screening. Excess unlabelled ovalbumin was also added to the library to prevent the isolation of ovalbumin binding cells.
In all, two rounds of MCS and two rounds of FACS with all three antigens were used to screen the library for binders. A portion of the remaining library cells was then screened by MCS with a single antigen and eight individual clones were isolated. The affinity of these clones was determined and the scFv region of one clone was sequenced. Despite preventative measures, all eight clones isolated and analyzed were found to have an affinity to undetermined ovalbumin complex regions other than the peptides of interest. Still, cells expressing scFvs binding to a portion of the antigen complexes presented to the library were clearly enriched and subsequently isolated using MCS and FACS.

Des études précédentes sur la levure Saccharomyces cerevisiae ont proposé une vacuolaire localisation pour Ath1, qui est difficile à concilier avec sa capacité à hydrolyser tréhalose exogènes. Dans notre étude, nous avons utilisé la microscopie fluorescente à montrer que Ath1 est bien localisées dans la vacuole, mais aussi à la surface cellulaire. Néanmoins, que Ath1 à la surface cellulaire est responsable pour la croissance sur tréhalose, et Ath1 dans la vacuole est enclin à protéolyse. Deuxième partie sur l’étude de domaine protéique, nous avons montré que les N-terminales 131 acides aminés de Ath1 sont le domaine potentiel pour l’adressage à la surface, parmi le domaine transmembranaire est indispensable. Enfin, la voie de ciblage de Ath1 dans la cellule de levure a été étudié. En utilisant différent mutants impliqués dans la voie de ciblage à la vacuole, nous avons prouvé que le ciblage d'Ath1 vers la vacuole emprunte une voie intracellulaire, indépendant de l’endocytosis. De plus, le ciblage à la surface probablement emprunte la voie "classique" de sécrétion en aide d’un group de Sec protéines. Ces études sont en cours
Trehalose (alpha-D-glucopyranosyl (1→1) alpha-D-gluocopyranoside) is a non-reducing disaccharide found in many organisms including yeasts, fungi, bacteria, plants and insects. In the yeast Saccharomyces cerevisiae, trehalose is one of the major storage carbohydrates, accounting for more than 25% of cell dry mass depending on growth conditions and stage of the yeast life cycle (Hottiger et al. , 1987a; Jules et al. , 2008; Lillie and Pringle, 1980). The accumulation of intracellular trehalose has two potential functions. First, it constitutes an endogenous storage of carbon and energy during spore germination and in resting cells. Second, trehalose acts as a stabilizer of cellular membranes and proteins (Francois and Parrou, 2001; Simola et al. , 2000; Singer and Lindquist, 1998). In the yeast S. Cerevisiae, trehalose is hydrolyzed into glucose by the action of two types of trehalases: the ‘neutral trehalases’ encoded by NTH1 and NTH2 (Jules et al. , 2008; Mittenbuhler and Holzer, 1988), which are optimally active at pH 7, and the ‘acid trehalase’ encoded by ATH1, showing optimal activity at pH 4. 5 (Destruelle et al. , 1995). Neutral trehalase has been well studied and is known to hydrolyze trehalose in the cytosol. While fungal acid trehalases, including the yeast Candida albicans (Pedreno et al. , 2004) and Kluyveromyces lactis (Swaim et al. , 2008) enzymes, have been reported to be localized at the cell surface, the localization of the S. Cerevisiae acid trehalase is still a matter of controversy. In 1982, Wiemken and coworkers (Keller et al. , 1982) first identified this protein in vacuole-enriched fraction obtained by density gradient centrifugation of a yeast protoplast preparation. Vacuolar localization of acid trehalase was very recently supported by in vivo imaging analyses using GFP-Ath1 fusion constructs under the strong and constitutive TPI1 promoter (Huang et al. , 2007). Furthermore, these authors employed various trafficking mutants to show that this acid trehalase reaches its vacuolar destination through the multivesicular body (MVB) pathway. However, this localization contrasts with the fact that this enzyme allows yeast to grow on exogenous trehalose (Nwaka et al. , 1995b), and with a measurable Ath1 activity at the cell surface (Jules et al. , 2004)

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.

In numerous Gram-negative and Gram-positive bacteria as well as in Archaea SL proteins form the outermost layer of the cell envelope. SL (glyco)monomers self-assemble with oblique (p2), tetragonal (p4), or hexagonal (p3, p6) symmetries [12]. SL subunits interact with each other and with the underlying cell surface by relatively weak non-covalent forces such as hydrogen-bonds, ionic bonds, salt-bridges or hydrophobic interactions. This makes them easy to isolate by applying chaotropic agents like urea and guanidine hydrochloride (GuHCl), chelating chemicals, or by changing the pH of the environment [10]. Upon dialysis in an ambient buffer monomers recrystallize into regular arrays that possess the forms of flat sheets, open ended cylinders, or spheres on solid substrates, at air-water intefaces and on lipid films, making them appealing for nanobiotechnological applications [3, 18]. The aim of this study was to investigate the structure, thermal stability, in vivo self-assembly process, recrystallization and metallization of three different recombinant SL proteins (SslA-eGFP, mSbsC-eGFP and S13240-eGFP) expressed in yeast S. cerevisiae BY4741 which could be further used in nanobiotechnological applications. In order to fulfill this aim, I investigated the in vivo expression of SL proteins (SslA, SbsC, S13240) tagged with eGFP (SL-eGFP) in the yeast S. cerevisiae BY4141. First, I characterized the heterologous expression of SL fusion constructs with growth and fluorescence measurements combined with Western blot analyses. Fluorescence microscopy investigations of overnight grown cultures showed that SslA-eGFP fusion protein was expressed as fluorescent patches, mSbsC-eGFP as tubular networks, and S13240-eGFP as hollow-like fibrillar network structures, while eGFP did not show any distinct structure Thermal stability of in vivo expressed SL-eGFP fusion proteins were investigated by fluorescence microscopy and immunodetection. In vivo self-assembly kinetics during mitosis and meiosis was the second main issue. In parallel, association of in vivo mSbsC-eGFP structures with the cellular components was of interest. A network of tubular structures in the cytosol of the transformed yeast cells that did not colocalize with microtubules or the actin cytoskeleton was observed. Time-resolved analysis of the formation of these structures during vegetative growth and sporulation was investigated by live fluorescence microscopy. While in meiosis ascospores seemed to receive assembled structures from the diploid cells, during mitosis surface layer structures were formed de novo in the buds. Surface layer assembly always started with the appearance of a dot-like structure in the cytoplasm, suggesting a single nucleation point. In order to get these in vivo SL assemblies stably outside the cells (in situ), cell distruption experiments were conducted. The tubular structures formed by the protein in vivo were retained upon bursting the cells by osmotic shock; however their average length was decreased. During dialysis, monomers obtained by treatment with chaotropic agents recrystallized again to form tube-like structures. This process was strictly dependent on calcium ions, with an optimal concentration of 10 mM. Further increase of the Ca2+ concentration resulted in multiple non-productive nucleation points. It was further shown that the lengths of the S-layer assemblies increased with time and could be controlled by pH. After 48 hours the average length at pH 9.0 was 4.13 µm compared to 2.69 µm at pH 5.5. Successful chemical deposition of platinum indicates the potential of recrystallized mSbsC-eGFP structures for nanobiotechnological applications. For example, such metalized protein nanotubes could be used in conductive nanocircuit technologies as nanowires.

Thermal stability is an important and interesting physical property of proteins. A common method to study it by is circular dichroism (CD) spectroscopy. The aim of this study was to test methods to improve thermal stability analysis by CD spectroscopy. Experiments were performed using the Abp1p SH3 domain from yeast as a model protein. Thermal denaturation was monitored at multiple wavelengths. It was concluded that for data sets of reasonable quality the choice of wavelength does not affect the results. An approach to estimate stability of thermophilic proteins was tested where thermal stability was measured at different concentrations of the denaturant GuHCl. The thermochemical data was used to estimate the stability in absence of GuHCl by extrapolation. The results were compared to those obtained from CD spectroscopy and differential scanning calorimetry. It was found that a stabilizing effect from low concentrations of GuHCl complicated the extrapolation. It is likely that this method is more successful if there is no stabilizing effect. The effect of ΔCp in stability parameter calculations was investigated with an experimentally and theoretically determined ΔCp. This was further investigated with synthetic data sets. The ΔCp used in calculations had no notable effect, as long as there was no cold denaturation. Although ΔCp is not necessary in calculations, it is an interesting parameter itself. ΔCp can be calculated from the thermochemical data used for extrapolation. The results in this study demonstrate robustness in thermal stability analysis by CD spectroscopy and a potential for development.

We report on the spatial and temporal signaling properties of a yeast pheromone-based cell communication and amplifier system. It utilizes the Saccharomyces cerevisiae mating response pathway and relies on diffusion of the pheromone α–factor as key signaling molecule between two cell types. One cell type represents the α–factor secreting sensor part and the other the reporter part emitting fluorescence upon activation. Although multi-cellular signaling systems promise higher specificity and modularity, the complex interaction of the cells makes prediction of sensor performance difficult. To test the maximum distance and response time between sensor and reporter cells, the two cell types were spatially separated in defined compartments of agarose hydrogel (5 ´ 5 mm) and reconnected by diffusion of the yeast pheromone. Different ratios of sensor to reporter cells were tested to evaluate the minimum amount of sensor cells required for signal transduction. Even the smallest ratio, one α–factor-secreting cell to twenty reporter cells, generated a distinct fluorescence signal. When using a 1:1 ratio, the secreted pheromone induced fluorescence in a distance of up to four millimeters after six hours. We conclude from both our experimental results and a mathematical diffusion model that in our approach: (1) the maximum dimension of separated compartments should not exceed five millimeters in gradient direction; and (2) the time-limiting step is not diffusion of the signaling molecule but production of the reporter protein.

We report on the spatial and temporal signaling properties of a yeast pheromone-based cell communication and amplifier system. It utilizes the Saccharomyces cerevisiae mating response pathway and relies on diffusion of the pheromone α–factor as key signaling molecule between two cell types. One cell type represents the α–factor secreting sensor part and the other the reporter part emitting fluorescence upon activation. Although multi-cellular signaling systems promise higher specificity and modularity, the complex interaction of the cells makes prediction of sensor performance difficult. To test the maximum distance and response time between sensor and reporter cells, the two cell types were spatially separated in defined compartments of agarose hydrogel (5 ´ 5 mm) and reconnected by diffusion of the yeast pheromone. Different ratios of sensor to reporter cells were tested to evaluate the minimum amount of sensor cells required for signal transduction. Even the smallest ratio, one α–factor-secreting cell to twenty reporter cells, generated a distinct fluorescence signal. When using a 1:1 ratio, the secreted pheromone induced fluorescence in a distance of up to four millimeters after six hours. We conclude from both our experimental results and a mathematical diffusion model that in our approach: (1) the maximum dimension of separated compartments should not exceed five millimeters in gradient direction; and (2) the time-limiting step is not diffusion of the signaling molecule but production of the reporter protein.

L'expression génétique est un processus cellulaire fondamental réglé de manière ne. Les promoteurs inducibles perme ent de perturber l'expression génétique en changeant l'expression d'une protéine par rapport à son niveau physiologique de référence. Ce e propriété en fait un outil incontournable pour décrypter le fonctionnement des processus biologiques via la comparaison du comportement de la cellule sous divers niveaux d'induction. Toutefois, une limite actuelle à l'utilisation des promoteurs inducibles provient de la difficulté à appliquer des perturbations précises et dynamiques. Les deux obstacles principaux étant: (i) la variabilité intercellulaire ainsi qu'à la nature aléatoire de l'expression génétique qui limite la précision de la perturbation appliquée. (ii) la difficulté à prèdire quantitativement le comporteement des systèmes biologiques sur les longues periodes requises pour des objectifs d'expression variables dans le temps. Or des perturbations précises et changeant dans le temps perme ent d'obtenir de riches informations sur la dynamique d'un système biologique. Est présenté ici une plate-forme de contrôle temps réel en boucle fermée qui permet le contrôle quantitatif sur une longue durée de l'expression génétique chez la levure. Ce e plateforme utilise la microscopie par uorescence pour suivre l'expression génétique, un système micro uidique pour interagir avec l'environnement cellulaire ainsi qu'un logiciel perme ant l'analyse d'image en temps réel et le calcul de la stratégie de contrôle à appliquer. Ce système permet le contrôle de l'expression d'un gène chez la levure, tant au niveau d'un population cellulaire qu'au niveau de la cellule seule et ceci pour un objectif d'expression constant ou dépendant du temps. Le système de réponse au chocs hyper-osmotiques de la levure S. cerevisiae (HOG pathway) a été utilisé pourin uencer l'expression génétique. Toutefois, la possibilité d'utiliser un autre système d'induction sans profondes modi cations de la plate-forme est démontrée. De surcroît au développement de ce e plate-forme est également ici démontré la possibilité de contrôler le système HOG. A n de comprendre la dynamique cellulaire et de pouvoir la quanti er, il est nécessaire de pouvoir appliquer des perturbations précises. La plate-forme de contrôle de l'expression génétique présentée ici permet de perturber avec précision le niveau d'expression d'une protéine et représente donc une contribution majeure dans ce e direction.

The aim of the theses was determination of selenium species in yeast Saccharomyces cerevisiae cultivated in medium with added inorganic form of selenium (Sodium Selenite). Concentrations of Sodium Selenite in cultivation medium were 0,1; 1; 10 and 100 mg.l-1. Cultivation was undertaken in fermenting tub for period of 72 hours. Cultivated yeasts were extracted by use of enzymes and subsequently the species of selenium in particular parts of yeasts were determined. In order to determine selenium species, the method of high-performance liquid chromatography in combination with atomic fluorescent spectrometer and technique of hydride generation was used. Having analysed different fractions of the yeasts Saccharomyces cerevisiae it was ascertained that during cultivation the sorption of selenium occurred in form of Se4+ in cell membranes while in cytoplasm no inorganic forms of selenium were found. Furthermore, it was stated that yeasts Saccharomyces cerevisiae are able to metabolically change inorganic forms of selenium to organic forms (selenomethionine), while these forms are present in cytoplasm and they are likely to be bound to proteinic structures of cell membranes. An increase of concentration of Se4+ in cell membranes could be observed as a result of increasing concentration of Sodium Selenite in cultivation medium. In proteinic structures the concentration of organic selenium forms increased only to concentration 10 mg.l-1 of Sodium Selenite in cultivation medium.

La membrane bactérienne a fait l'objet de nombreuses études de localisation de protéines et de phospholipides. Par fusion d’une protéine fluorescente (GFP) aux gènes d’intérêt, il est alors possible d‘observer la localisation des protéines associées par microscopie. La plupart de ces observations ont été réalisées à l’aide de microscopes dits à épifluorescence. Afin d’obtenir une qualité d’image suffisante, il était nécessaire de surexprimer la protéine observée, insérée à un locus ectopique non naturel. Ce travail de thèse a permis d’utiliser une nouvelle technologie acquise dans notre laboratoire, le microscope à fluorescence par réflexion totale interne (TIRFM), plus puissant que le microscope à épifluorescence utilisé précédemment. Cette technologie a permis une caractérisation plus détaillée de la localisation de protéines d’intérêt, placées sous contrôle de leur promoteur naturel. Il a également été possible de caractériser la dynamique des foci observés. Nous avons concentré notre étude sur 3 protéines: (i) SecA pour l’étude de la translocation des protéines du cytoplasme vers la membrane, (ii) YidC pour l’insertion des protéines dans la membrane, (iii) PgsA pour la synthèse des phospholipids. Les foci se déplacent dynamiquement et s’associent de manière transitoire dans la membrane. L’observation sur la durée de ces foci, et l’analyse de leur intensité moyenne au cours des observations, montre que SecA se déplace sur l’ensemble de la membrane de manière uniforme. L’analyse du déplacement des foci montre une relation quadratique entre la distance moyenne parcourue par les foci en fonction du temps. Ce résultat est en accord avec l’hypothèse d’un mouvement brownien des foci. Les foci sont observés dans les différentes phases de croissance des cellules, et le nombre de foci présents dans une cellule de la longueur de celle-ci. SecA-GFP a été testés dans un certain nombre de contextes génétiques. La localisation a été perturbée lors de la déplétion de pgsA. Cependant, comme PgsA est une protéine essentielle, il ne peut être exclu que ce changement de localisation apparaît des cellules qui sont mortes ou mourantes. Dans une souche mutante ΔclsA, on n’observe aucune différence dans la localisation de SecA en phase exponentielle, mais on aperçoit une relocalisation aux poles en phase stationnaire de croissance. La voie Tat est responsable du transport des protéines devant être exportées dans un état structuré, par exemple dans le cas de l’incorporation d’un co-facteur. À ce jour, la régulation du système Tat est peu connues, de même que les interactions entre les différentes sous-unités du système Tat et d'autres protéines dans le cytoplasme, dans la membrane ou dans la paroi cellulaire. Des fusions de les gènes de la voie Tat ont été co-exprimées deux à deux dans des cellules de levure, et leur capacité à interagir in vivo a été testée par la méthode dite du double hybride chez la levure. Nous avons généré un réseau d’interaction autour des cinq composants de système Tat. Pour déterminer les implications fonctionnelles des composants du réseau, nous avons travaillé en collaboration avec le laboratoire de Jan-Maarten van Dijl. Nous avons utilisé une collection de souches mutantes pour lesquels certains composants individuels du réseau ont été retires. Trois a été observe d’etre nécessaires pour la sécrétion Tat-dépendante. Nous avons étudié la localisation des fusions GFP avec ces proteins. On a observé une localisation double de HemAT selon l’état physiologique de la cellule. En phase exponentielle, les cellules de B. subtilis sont généralement présentes sous forme de chaînes dans lesquelles le septum de division a déjà été formé, mais la séparation cellulaire n'a pas encore eu lieu. Une fusion de la GFP à CsbC apparaît de façon homogène dans la membrane
In the years since the cloning of GFP, the field of bacterial cell biology has characterized a variety of specific protein localization patterns in the bacterial membrane. The vast majority of early subcellular localization studies made use of inducible GFP fusions, which generally required the presence of high concentrations of inducer, and can therefore be considered to be overexpressed. An outstanding question remains over the organization of natively expressed proteins in the membrane. Here, we have investigated the localization of functional GFP fusions to proteins catalyzing important membrane processes; the secretion motor protein SecA, the membrane insertase YidC1, and the essential phospholipid synthase PgsA using total internal reflection fluorescence microscopy (TIRFM). This allowed natively expressed proteins to be localized with temporal resolution that can capture their dynamics. We characterized dynamic complexes dispersed throughout the membrane displaying diffusive movement with no preferred trajectories. Further characterization focused upon identifying conditions in which the localization pattern was disturbed. A polar mislocalization was identified in a cardiolipin mutant strain. The yeast two-hybrid (Y2H) approach is a robust approach to detect binary interactions on a proteome-scale. We performed genome-wide Y2H screens as well as targeted Y2H analyses for specific interactions involving components of the Sec and Tat secretion machineries of B. subtilis, revealing an intricate protein-protein interaction network involving 71 proteins. Furthermore, three proteins identified in the Tat network, WprA, CsbC and HemAT, were shown to be important for effective protein secretion via the B. subtilis Tat system, indicating that our yeast two hybrid assays reveal biologically significant interactions involving membrane proteins. The studies provide a novel proteomic view on the interaction network of the secretion systems of B. subtilis

Parmi les espèces de levures du clade CTG, certaines sont responsables de candidoses tandis que d’autres présentent des potentiels en biotechnologie. Depuis quelques années, nous assistons à une intensification des recherches sur ces levures. Cependant, leur code génétique particulier a ralenti la mise au point d’outils génétiques pour la plupart d’entre elles. Cette thèse vise à développer des outils moléculaires standardisés pour un grand nombre d’espèces de levures du clade CTG. Nous avons d’abord conçu des vecteurs d’expression adaptés à l’espèce M. guilliermondii. Par la suite, nous avons caractérisé le gène de résistance à l’acide mycophénolique IMH3.2 afin de l’utiliser comme marqueur de sélection lors de la transgénèse d’espèces du clade CTG. Enfin, nous avons mis au point une série de vecteurs permettant la manipulation génétique de ces espèces. Ce travail a conduit à la conception d’une large gamme d’outils utilisable dans un grand nombre de ces levures, pré-requis essentiel aux futurs recherches en mycologie médicale et au développement de stratégies de biologie synthétique
The fungal CTG clade includes well-known yeasts of clinical importance and/or biotechnological potential. Thus, albeit being intensively studied over the last 30 years, their uncommon genetic code precludes the use of the widely available markers and reporter systems for genetic approaches in these microorganisms. We provide here a toolbox to genetically manipulate a wide range of CTG clade species. Firstly, we developed a new series of versatile controllable expression vectors for M. guilliermondii. After, we characterized MPA-resistant gene IMH3.2 et used it as a drug resistance marker in several yeast species. Finaly, we provide a molecular toolbox suitable to genetically manipulate a broad range of prominent species from the CTG clade. This versatile toolkit represents a new starting point for successful developments of research in medical mycology in the CTG clade but also will expedite synthetic biology strategies in these microorganisms for biotechnological applications

Thesis (MTech (Food Technology)--Cape Peninsula University of Technology, 2009
The primary aim of this study was to establish the effects of both cavitation, (20 KHZ), and heat (55°C or 60•C) on Saccharomyces cerevisiae GC210 (MATa lys2) suspended in physiological saline. Fluorescent flow cytometry was used to determine the effects of moist heat and acoustic cavitation on S. cerevisiae cells. Results from this study could be used as a guide for use by the food industry for the combined use of heat and sonication to disinfect various solutions contaminated with S. cerevisiae.

Understanding the functional impact of physical interactions between proteins and DNA on gene expression is important for developing approaches to correct disease-associated gene dysregulation. I conducted a systematic, functional genetic analysis of protein-DNA interactions in the promoter region of the yeast ribonucleotide reductase subunit gene RNR3. I measured the transcriptional impact of systematically perturbing the major transcriptional regulator, Crt1, and three X-box sites on the DNA known to physically bind Crt1. This analysis revealed interactions between two of the three X-boxes in the presence of Crt1, and unexpectedly, a significant functional role of the X-boxes in the absence of Crt1. Further analysis revealed Crt1- independent regulators of RNR3 that were impacted by X-box perturbation. Taken together, these results support the notion that higher-order X-box-mediated interactions are important for RNR3 transcription, and that the X-boxes have unexpected roles in the regulation of RNR3 transcription that extend beyond their interaction with Crt1.

[EN] ABSTRACT Abscisic acid (ABA) signaling plays a critical role in regulating root growth and root system architecture. ABA-mediated growth promotion and root tropic response under water stress are key responses for plant survival under limiting water conditions. In this work, we have explored the role of Arabidopsis (Arabidopsis thaliana) PYR/PYL/RCAR receptors (PYRABACTIN RESISTANCE1 (PYR1)/PYR1 LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS) for root ABA signaling. As a result, we discovered that PYL8 plays a nonredundant role for the regulation of root ABA sensitivity. Unexpectedly, given the multigenic nature and partial functional redundancy observed in the PYR/PYL family, the single pyl8 mutant showed reduced sensitivity to ABA-mediated root growth inhibition. This effect was due to the lack of PYL8-mediated inhibition of several clade A phosphatases type 2C (PP2Cs), since PYL8 interacted in vivo with at least five PP2Cs, namely HYPERSENSITIVE TO ABA1 (HAB1), HAB2, ABAINSENSITIVE1 (ABI1), ABI2, and PP2CA/ABA-HYPERSENSITIVE GERMINATION3 as revealed by tandem affinity purification and mass spectrometry proteomic approaches. Membrane-delimited abscisic acid (ABA) signal transduction plays a critical role in early ABA signaling, but the molecular mechanisms linking core signaling components to the plasma membrane are unclear. We show that transient calciumdependent interactions of PYR/PYL/RCAR ABA receptors with membranes are mediated through a 10-member family of C2-domain ABA-related (CAR) proteins in Arabidopsis thaliana. Specifically, we found that PYL4 interacted in an ABA-independent manner with CAR1 in both the plasma membrane and nucleus of plant cells. CAR1 belongs to a plant-specific gene family encoding CAR1 to CAR10 proteins, and bimolecular fluorescence complementation and coimmunoprecipitation assays showed that PYL4-CAR1 as well as other PYR/PYL-CAR pairs interacted in plant cells. The crystal structure of CAR4 was solved, which revealed that, in addition to a classical calcium-dependent lipid binding C2 domain, a specific CAR signature is likely responsible for the interaction with PYR/PYL/RCAR receptors and their recruitment to phospholipid vesicles. This interaction is relevant for PYR/PYL/RCAR function and ABA signaling, since different car triple mutants affected in CAR1, CAR4, CAR5, and CAR9 genes showed reduced sensitivity to ABA in seedling establishment and root growth assays. In summary, we identified PYR/PYL/RCAR-interacting partners that mediate a transient Ca2+-dependent interaction with phospholipid vesicles, which affects PYR/PYL/RCAR subcellular localization and positively regulates ABA signaling.
[ES] RESUMEN La señalización por la hormona vegetal ácido abscísico (ABA) desempeña un papel crítico en la regulación del crecimiento de la raíz y en la arquitectura del sistema radical. La promoción de crecimiento de la raíz en condiciones de estrés hídrico mediada por ABA es clave para la supervivencia de las plantas bajo condiciones limitantes de agua. En este trabajo, hemos explorado el papel de los receptores PYR/PYL/RCAR (PYRABACTIN RESISTANCE1 (PYR1)/PYR1 LIKE (PYL)/ REGULATORY COMPONENTS OF ABA RECEPTORS) de Arabidopsis (Arabidopsis thaliana) en la ruta de señalización de ABA en raíz. Así, hemos descubierto que el receptor de ABA PYL8 juega un papel no redundante en la regulación de la percepción de ABA en raíz. Inesperadamente, dada la naturaleza multigénica y la redundancia funcional parcial observada en la familia PYR/PYL/RCAR, el mutante pyl8 fue el único mutante sencillo de pérdida de función de los receptores PYR/PYL/RCAR que mostraba una sensibilidad reducida a la inhibición del crecimiento mediada por ABA en raíz. Este efecto se debe a la falta de inhibición mediada por PYL8 de varias fosfatasas del grupo A tipo 2C (PP2Cs), ya que PYL8 es capaz de interactuar in vivo con al menos cinco PP2Cs, denominadas HYPERSENSITIVE TO ABA1 (HAB1), HAB2, ABAINSENSITIVE1 (ABI1), ABI2, and PP2CA/ABA-HYPERSENSITIVE GERMINATION3 según lo han revelado la purificación por afinidad en tándem (TAP por sus siglas en inglés) y estudios proteómicos de espectrometría de masas. La transducción de la señal del ABA localizada en la membrana plasmática celular juega un papel crucial en los pasos iniciales de la señalización de la fitohormona, pero los mecanismos moleculares que unen los componentes básicos de la señalización y la membrana plasmática no están claros. Estudiando las interacciones de los receptores del ABA PYR/PYL/RCAR con la membrana plasmática hemos encontrado que éstos pueden interaccionar transitoriamente con ella de forma dependiente de calcio gracias a una familia de proteínas con dominios C2 relacionadas con la ruta de señalización de ABA (denominadas C2-domain ABA-related (CAR) proteins). Específicamente, se encontró que PYL4 interacciona de manera independiente de ABA con CAR1 tanto en la membrana plasmática como en el núcleo de las células vegetales. La proteína CAR1 pertenece a una familia multigénica constituida por 10 miembros en Arabidopsis thaliana, desde CAR1 hasta CAR10, y que solo se encuentra en plantas. Los ensayos de complementación bi-molecular de fluorescencia y de co-immunoprecipitación confirmaron la interacción en células vegetales tanto de PYL4-CAR1 como de otras parejas de PYR/PYL-CAR. La cristalización de la proteína CAR4 reveló que, además de un dominio C2 clásico de unión a lípidos dependiente de calcio, las proteínas de la familia CAR presentan un dominio específico que probablemente es responsable de la interacción con los receptores PYR/PYL/RCAR y de su posterior reclutamiento a las vesículas de fosfolípidos. Esta interacción es relevante para la función de los receptores PYR/PYL/RCAR en la señalización del ABA, ya que diferentes mutantes triples car de pérdida de función, que tienen afectados los genes CAR1, CAR4, CAR5, y CAR9, demostraron una reducción de la sensibilidad al ABA en ensayos de establecimiento de plántula y crecimiento de la raíz. En resumen, hemos identificado nueva familia de proteínas que son capaces mediar las interacciones transitorias dependientes de Ca2+ con vesículas de fosfolípidos, lo que a su vez afecta localización de PYR/PYL/RCAR y regula positivamente la señalización de ABA.
[CAT] RESUM La senyalització per l'hormona vegetal àcid abcíssic (ABA) exerceix un paper crític en la regulació del creixement de l'arrel i també en l'arquitectura del sistema radical. La promoció del creixement de l'arrel en condicions d'estrés hídric, regulada per ABA és clau per la supervivència de les plantes sota condicions limitants d'aigua. Amb aquest treball, hem investigat el paper dels receptors PYR/PYL/RCAR (PYRABACTIN RESISTANCE1 (PYR1)/PYR1 LIKE (PYL)/ REGULATORY COMPONENTS OF ABA RECEPTORS) d'Arabidopsis (Arabidopsis thaliana) en el camí de senyalització d'ABA en arrel. Així, hem descobert que el receptor d'ABA PYL8 exerceix un paper no redundant en la regulació de la percepció d'ABA en arrel. Inesperadament, donada la naturalesa multigènica i la redundància funcional parcial que s'observa en la família PYR/PYL/RCAR, el mutant pyl8 va ser l'únic mutant senzill de pèrdua de funció dels receptors PYR/PYL/RCAR que mostrava una sensibilitat reduïda a la inhibició del creixement mitjançada per l'ABA en l'arrel. Doncs aquest efecte es deu a la falta d'inhibició regulada per PYL8 de diverses fosfatases del grup A tipus 2C (PP2Cs), ja que PYL8 té la capacitat d'interactuar in vivo almenys amb cinc PP2Cs, anomenades HYPERSENSITIVE TO ABA1 (HAB1), HAB2, ABAINSENSITIVE1 (ABI1), ABI2, and PP2CA/ABAHYPERSENSITIVE GERMINATION3 segons ho han revelat per una banda la purificació per afinitat en tàndem (TAP són les seues sigles en anglés) i per altra banda, estudis proteòmics d'espectrometria de masses. Pel que fa a la transducció del senyal del l'ABA, la qual es localitza en la membrana plasmàtica cel¿lular, juga un paper molt important en els primers instants de la senyalització de la fitohormona, no obstant això els mecanismes moleculars que uneixen els components bàsics d'aquesta senyalització amb la membrana plasmàtica, no es troben del tot clars. Per tant, s'han estudiat les interaccions que tenen els receptors del ABA PYR/PYL/RCAR amb la membrana plasmàtica, i hem trobat que aquests tenen la capacitat d'interaccionar transitòriament amb la membrana de forma dependent al calci, gràcies a una família de proteïnes amb domini C2, les quals es troben relacionades amb la ruta de senyalització d'ABA(anomenades C2domain ABArelated (CAR) proteins).Específicament, es va trobar que PYL4 interacciona d'una manera independent al ABA amb CAR1, tant en la membrana plasmàtica, com en el nucli de les cèl¿lules vegetals. La proteïna CAR1 pertany a la família multigènica constituïda per 10 components en Arabidopsis thaliana, des de CAR1 fins CAR10, que tan sols es troba en plantes. Els assajos de complementació bimolecular de fluorescència i de co-immunoprecipitació, van confirmar la interacció en cèl¿lules vegetals, tant de PYL4CAR1 com d'altres parelles de PYR/PYL-CAR. La cristal¿lització de la proteïna CAR4 va revelar que, a més d'un domini C2 clàssic de unió a lípids dependent del calci, les proteïnes de la família CAR presenten un domini PYR/PYL/RCAR, i del seu posterior reclutament a les vesícules fosfolipídiques. Doncs, aquesta interacció és rellevant en la funció dels receptors PYR/PYL/RCAR, ja que participa en la senyalització del l'ABA. Aquesta interacció es clau per a la funció dels receptors, ja que diferents mutants triples car de pèrdua de funció, els quals posseïxen afectats els gens CAR1, CAR4, CAR5 i CAR9, van mostrar una reducció de la sensibilitat a l'ABA en assajos d'establiment de plàntula i creixement de l'arrel. En conclusió, hem identificat una nova família de proteïnes amb la capacitat d'organitzar les interaccions transitòries dependents del calci amb vesícules de fosfolípids, fet que al seu torn afecta la localització de PYR/PYL/RCAR i regula positivament la senyalització d'ABA.
Rodríguez Solovey, LN. (2015). IDENTIFICATION OF TARGETS AND AUXILIARY PROTEINS OF PYR/PYL/RCAR ABA RECEPTORS: PROTEIN PHOSPHATASES TYPE 2C (PP2Cs) AND C2-DOMAIN ABA-RELATED PROTEINS (CARs) [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/58862
TESIS

The field of metabolic engineering focuses on using molecular biology tools to genetically modify the metabolic pathways of cells for the production of chemical compounds. The field of directed evolution can alter the native abilities of proteins by taking inspiration from natural evolution. Both fields bring novel solutions to current problems in energy, the environment, and medicine. However, there is still no general higher throughput screening method for both of these fields. In this dissertation, we apply our designed fluorescence polarization assay to fill this need in the fields of metabolic engineering and directed evolution. Chapter 0 gives background information related to metabolic engineering, directed evolution, tetracyclines, the Tetracycline Repressor protein (TetR), TAN-1612, and fluorescence polarization. Chapter 1 describes our development of a quantitive, sensitive, and fast fluorescence polarization assay which uses the TetR protein to detect the binding of the small molecule tetracycline TAN-1612. Chapter 2 demonstrates that the binding affinity of the TetR protein for TAN-1612 can be improved using directed evolution and by incorporating our assay to screen TetR mutants. Finally, in Chapter 3 we apply our fluorescence polarization assay to the screening of yeast strains biosynthesizing TAN-1612, without the need of time and labor intensive extraction and purification steps.

Title: Changes of intracellular pH in yeast cells under stress conditions Author: Radek Divín Department: Institute of Physics of Charles University Supervisor: prof. RNDr. Jaromír Plášek, CSc. Abstract: Specific values of intracellular pH (pHi) can affect all biochemical processes in a cell and this phenomenon is closely connected with the degree of importance of changes in the intracellular pH under the stress conditions. In the Master Thesis, the yeast cells Saccharomyces cerevisiae were used as a model of organism eukaryotic cells. Monitoring of intracellular pH of the cells was performed by the method of synchronous fluorescence scan technique of genetically encoded fluorescent probes pHluorin which was located in the cytosol of the cells. The cells were exposed to stress conditions due to the chemical changes in the environment. Consequently, their ability to maintain a stable value of the intracellular pH in various acidic environments was studied in more detail. The attention was also focused on the impact on optimizing of glucose cytosolic pH. The work was centered on the changes in intracellular pH under the influence of the presence of KCl in suspension. Furthermore, the decrease of cytosolic pH of monitored cells by protonophore CCCP was investigated. The effect of stress environment on the...

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.

In numerous Gram-negative and Gram-positive bacteria as well as in Archaea SL proteins form the outermost layer of the cell envelope. SL (glyco)monomers self-assemble with oblique (p2), tetragonal (p4), or hexagonal (p3, p6) symmetries [12]. SL subunits interact with each other and with the underlying cell surface by relatively weak non-covalent forces such as hydrogen-bonds, ionic bonds, salt-bridges or hydrophobic interactions. This makes them easy to isolate by applying chaotropic agents like urea and guanidine hydrochloride (GuHCl), chelating chemicals, or by changing the pH of the environment [10]. Upon dialysis in an ambient buffer monomers recrystallize into regular arrays that possess the forms of flat sheets, open ended cylinders, or spheres on solid substrates, at air-water intefaces and on lipid films, making them appealing for nanobiotechnological applications [3, 18]. The aim of this study was to investigate the structure, thermal stability, in vivo self-assembly process, recrystallization and metallization of three different recombinant SL proteins (SslA-eGFP, mSbsC-eGFP and S13240-eGFP) expressed in yeast S. cerevisiae BY4741 which could be further used in nanobiotechnological applications. In order to fulfill this aim, I investigated the in vivo expression of SL proteins (SslA, SbsC, S13240) tagged with eGFP (SL-eGFP) in the yeast S. cerevisiae BY4141. First, I characterized the heterologous expression of SL fusion constructs with growth and fluorescence measurements combined with Western blot analyses. Fluorescence microscopy investigations of overnight grown cultures showed that SslA-eGFP fusion protein was expressed as fluorescent patches, mSbsC-eGFP as tubular networks, and S13240-eGFP as hollow-like fibrillar network structures, while eGFP did not show any distinct structure Thermal stability of in vivo expressed SL-eGFP fusion proteins were investigated by fluorescence microscopy and immunodetection. In vivo self-assembly kinetics during mitosis and meiosis was the second main issue. In parallel, association of in vivo mSbsC-eGFP structures with the cellular components was of interest. A network of tubular structures in the cytosol of the transformed yeast cells that did not colocalize with microtubules or the actin cytoskeleton was observed. Time-resolved analysis of the formation of these structures during vegetative growth and sporulation was investigated by live fluorescence microscopy. While in meiosis ascospores seemed to receive assembled structures from the diploid cells, during mitosis surface layer structures were formed de novo in the buds. Surface layer assembly always started with the appearance of a dot-like structure in the cytoplasm, suggesting a single nucleation point. In order to get these in vivo SL assemblies stably outside the cells (in situ), cell distruption experiments were conducted. The tubular structures formed by the protein in vivo were retained upon bursting the cells by osmotic shock; however their average length was decreased. During dialysis, monomers obtained by treatment with chaotropic agents recrystallized again to form tube-like structures. This process was strictly dependent on calcium ions, with an optimal concentration of 10 mM. Further increase of the Ca2+ concentration resulted in multiple non-productive nucleation points. It was further shown that the lengths of the S-layer assemblies increased with time and could be controlled by pH. After 48 hours the average length at pH 9.0 was 4.13 µm compared to 2.69 µm at pH 5.5. Successful chemical deposition of platinum indicates the potential of recrystallized mSbsC-eGFP structures for nanobiotechnological applications. For example, such metalized protein nanotubes could be used in conductive nanocircuit technologies as nanowires.

Title: Deconvolution fluorescence microscopy of yeast cells Author: Tomáš Štec Department: Institute of Physics of Charles University Supervisor: prof. RNDr. Jarmoír Plášek, CSc., Institute of Physics of Charles Uni- versity Abstract: Fluorescence microscopy presents an fast and cheap alternative to more advanced imaging methods like confocal and electron microscopy, even though it is subject to heavy image distortion. It is possible to recover most of the original distortion-free image using deconvolution in computer image processing. This al- lows reconstruction of 3D structure of studied objects. Deconvolution procedure of NIS Elements AR program undergoes an thorough inspection in this diploma the- sis. It is then applied on restoration of 3D structure of calcofluor stained cell wall of budding yeast Saccharomyces cerevisiae. Changes of the structure of the cell wall during cell ageing are being examined. Cell wall of aged cells shows increased surface roughness and even ruptures at the end of cell life. Keywords: fluorescence, microscopy, deconvolution, NIS Elements AR, calcofluor, yeast, cell wall, ageing

The master's thesis is focused on the study of response of the intracellular pH of the yeast cells on various external environments, primarily in a relation to the protonophore carbonyl cyanide m-chlorophenylhydrazone, CCCP. To measure the intracellular pH of the yeast cells we used a genetically coded fluorescent probe the ratiometric pHluorin. Using the method of synchronously scanned fluorescent spectra we were able to measure the intracellular pH of the cells with high precision. As a part of these experiments we also studied the influence of ionic strength of the cell suspensions buffers on the surface potential as well as the influence of the mineral salt KCl on the depolarization of the yeast membranes and cytosolic acidification induced by the protonophore CCCP. We examined the changes of cytosolic pH as such but we also used the measured pH as an indicator of the processes and the state of environment outside the cell. One of the most notable outcomes of this thesis is a new method of monitoring the value of the surface potential of the yeast cells by measuring the titration curves of cytosolic acidification induced by the protonophore CCCP.

As demand for food increases, rapid testing methods are becoming increasingly important. In the past few years, yogurt has become popular. Yeast species are the most common spoilage organism, costing consumers and food companies money. A novel lateral flow assay has been developed to detect yeast oligonucleotide sequences. Gold nanoparticles were used as the standard reporter and fluorescent nanoparticles were developed as the novel reporter. The fluorescent nanoparticles were ruthenium-doped silica nanoparticles synthesized using the modified Stöber method. Visual analysis of assays using standard reporters showed the limit of detection to be 10 femtomoles of target sequence. Analysis of the fluorescent nanoparticles using a plate reader showed the limit of detection to be 0.027 femtomoles. The fluorescent reporter’s limit of detection is 1000 fold lower due to a sophisticated, more sensitive analysis method. Gold nanoparticles are appropriate for presence or absence testing, but fluorescent nanoparticles are best for obtaining quantitative data with low detection limits. Pathogens have been used as biological warfare for centuries. A brief review of common biowarfare agents is included. Yersinia pestis, the causative agent of the Plague, and Bacillus anthracis, the causative agent of Anthrax, are the focus. Additional work using gold nanoparticles as reporter in a sandwich assay is also included. The novel dye covered reporter was compared to the control, which was a single dye molecule linked to the reporter sequence. Repeated testing showed the novel reporter had a lower limit of detection and higher sensitivity due to increased ability to bind target.

The membrane potential is one of the most important parameters of the living cell. It can be measured using carbocyanine fluorescent probes. In this thesis we examined parameters of several dyes of this family. For further experiments three of them were chosen - diOC3(3), diIC1(3) a diIC2(5) as a supplement to diSC3(3) and diSC3(5), which represent standard probes used at biophysical department of Institut of Physics. We compared the rates of their accumulation in S. cerevisiae cells to determine if they were MDR pumps' substrates. The other goal of this work was to decide whether the results obtained using different probes are equivalent and to determine if the presence of a probe affects the spectral characteristics of another. For this purpose we have chosen diSC3(3) and diSC3(5). With those dyes we examined the influence of the acidification on membrane potencial of the yeast S. cerevisiae. We showed that the information on depolarization obtained using both probes were matching very well.

In this thesis, emphasis has been placed on development of new approaches for high-throughput analysis of protein:DNA interactions in vitro and in vivo. In vitro strategies for detection of protein:DNA interaction require isolation of active and soluble protein. However, current methodologies for purification of proteins often fail to provide high yield of pure and tag-free protein mainly because enzymatic cleavage reactions for tag removal do not exhibit stringent sequence specificity. Solving this problem is an important step towards high-throughput in vitro analysis of protein:DNA interactions. As a result, parts of this thesis are devoted to developing new approaches to enhance the specificity of a proteolysis reaction. The first approach was through manipulation of experimental conditions to maximize the yield of the desired protein products from enterokinase proteolysis reactions of two His-tagged proteins. Because it was suspected that accessibility of the EK site was impeded, that is, a structural problem due to multimerization of proteins, focus was based on use of denaturants as a way to open the structure, thereby essentially increasing the stoichiometry of the canonical recognition site over noncanonical, adventitious sites. Promoting accessibility of the canonical EK target site can increase proteolytic specificity and cleavage yield, and general strategies promoting a more open structure should be useful for preparation of proteins requiring endoprotease treatment. One such strategy for efficient EK proteolysis is proposed: by heterodimerizing with a separate leucine zipper, the bZIP basic region and amino-terminus can become more open and potentially more accessible to enterokinase. In vivo strategies have the advantage over their in vitro counterparts of providing a native-like environment for assessing protein:DNA interactions, yet the most frequently used techniques often suffer from high false-positive and false-negative rates. In this thesis, a new bioprobe system for high-throughput detection of protein:DNA interactions in vivo is presented. This system offers higher levels of accuracy and sensitivity as well as accessibility and ease of manipulation in comparison with existing technologies.

The current increased use of antifungal agents has resulted in the development of resistance to these drugs. Search for new antifungals with different mechanisms of action overcoming the multidrug resistance is thus underway. Surface-active antifungals have the advantages of minimizing host toxicity and the emergence of drug resistance. We have developed a fluorescence method based on the use of the potentiometric fluorescent probe diS-C3(3), substrate of two major S. cerevisiae MDR pumps, Pdr5p and Snq2p. It allows us to monitor with high sensitivity and in real time changes in the activities of both pumps and also in membrane potential. We present here an efficient strategy for identifying pump inhibitors with minimal side effects on membrane integrity, and compare the potencies of different inhibitors towards MDR pumps. New efficient inhibitors of MDR pumps could potentially be used in conjunction with current antimicrobials that are MDR pump substrates. The method can be also used to determine the mechanism of action of surface-active drugs and their lowest effective concentrations.

Competition is a very important natural phenomenon, which causes the rivalry of organisms, in cases such as space limitation or lack of nutrients. It occurs mainly in situations where organisms, including microorganisms live in large populations. Multicellular yeast colonies represent an example of such a population. After the population of yeast cells spends nutrients from the environment, the cells in colonies are able to respond to these changes by production of ammonia functioning as a signaling molecule. Subsequently, the cells are able to change their morphology and metabolism and, dependently on their location within the colony, to create a subpopulation of cells with specific characteristics and functions. It is likely that in the case of mixed colonies formed by the two different strains, a competition between the cells of these two strains could exist. Such rivalry can result in changes in the ratio of cells of the two strains within the colony population, so that the cells of one strain outweigh the other. In this diploma thesis, I compared the growth and development of giant colonies and competition between the cells of selected pairs of strains forming mixed colonies. I focused on the parental strain Saccharomyces cerevisiae BY and its variants labeled with fluorescent proteins. For...