De nombreux processus physiologiques tels que les mouvements stomatiques, l’absorption des nutriments, l’élongation cellulaire et la signalisation cellulaire impliquent des flux d’anions entre les membranes plasmique et vacuolaire des cellules végétales. Ces flux ioniques sont régulés par des canaux et transporteurs membranaires. Les canaux ioniques transportent passivement les ions au travers des membranes selon le gradient électrochimique. Les transporteurs actifs permettent le transport contre le gradient électrochimique de l’ion transporté induisant son accumulation dans un compartiment cellulaire. Dans les cellules végétales, le gradient de H+ entre différents compartiments constitue la principale source d’énergie couplée par les symports et les antiports au transport de NO₃⁻ et Cl⁻. Au cours de ma thèse, j’ai analysé ces flux ioniques avec deux approches. Une première approche a consisté en l’étude fonctionnelle par électrophysiologie de deux protéines membranaires, AtCLCc et AtCLCg impliquées dans le transport d’anions. Dans une deuxième approche, un biosenseur, clopHensor a été exprimé chez A. thaliana et a permis de mesurer simultanément la [NO₃⁻] et le pH cytosoliques in vivo. Les cellules de garde ont été choisies comme modèle cellulaire pour l’étude de la dynamique in vivo de la [NO₃⁻]cyt et du pH. Nous avons mis en évidence que la [NO₃⁻]cyt est influencée par les conditions extracellulaires dans ces cellules. Enfin l’expression de clopHensor en plantes KO pour un antiport NO₃⁻/H⁺ vacuolaire, AtCLCa, et d’un canal anionique de la membrane plasmique, SLAC1, nous a permis d’étudier la contribution de deux membranes dans la régulation de [NO₃⁻] et du pH cytosolique. Les travaux menés ont permis de visualiser l’activité de canaux et de transporteurs d’anions et H⁺ in vivo et de quantifier leur impact sur l’homéostasie du cytosol. / Many physiological processes like stomata aperture, nutrient up-take, cellular elongation and cell signalling involve anion fluxes at the two main membranes, the plasma and vacuolar membranes of plant cells. Specialized membrane proteins form active and passive anion transport systems mediating and regulating anion fluxes. Ion channels are passive transport systems mediating ion fluxes across membranes along the electrochemical gradient. Whereas active transporters work against the electrochemical gradient of the transported ion allowing its accumulation into a cellular compartment. In plant cells, the H⁺ gradient is the main energy source of antiporters and symporters that couple the transport of anions like NO₃⁻ and Cl⁻ to the transport of H⁺. In the presents work, we aimed at analysing anion and H⁺ fluxes at two levels. First, we used an electrophysiological approach to study the functional properties of two anion transport systems acting at the vacuolar membrane, AtCLCc and AtCLCg. We also expressed a biosensor, clopHensor in A. thaliana to dynamically measure in vivo the [NO₃⁻] and pH of the cytosol. We chose stomata guard cells as a cellular model to study these fluxes. Our results illustrate the in vivo dynamics of cytosolic [NO₃⁻] and pH variations in the cytosol of guard cells. Our data show that in guard cells the cytosolic [NO₃⁻] is highly influenced by the extracellular [NO₃⁻]. At last, clopHensor’s expression in plants KO for the vacuolar NO₃⁻/H⁺ antiporter AtCLCa and for the plasma membrane anion channel SLAC1 allowed us to dissect the role of the two membranes in controlling the variation of cytosolic [NO₃⁻] and pH. This work enabled to visualize the activity of an anion channel (SLAC1) and of a NO₃⁻/H⁺ antiporter (AtCLCa) in vivo and to quantify the impact of anion and proton fluxes on cytosolic homeostasis of guard cells.

Low bioavailability of phosphate (P) due to low concentration and high immobility in soils is a key limiting factor in crop production. Application of excess amounts of P fertilizer is costly and by no means sustainable, as world-wide P resources are finite and running out. To facilitate the breeding of crops adapted to low-input soils, it is essential to understand the consequences of P deficiency. The second messenger calcium (Ca2+) is known to signal in plant development and stress perception, and most recently its direct role in signalling nutrient availability and deficiency has been partially elucidated. The use of Ca2+ as a signal has to be tightly controlled, as Ca2+ easily complexes with P groups and therefore is highly toxic to cellular P metabolism. It is unknown whether Ca2+ signals P availability or whether signalling is altered under P starvation conditions. The aim of this PhD project was to characterise the use of Ca2+ ions, particularly cytosolic free Ca2+ ([Ca2+]cyt), in stress signalling by P-starved roots of the model plant Arabidopsis thaliana. The hypothesis was that under P starvation and a resulting decreased cellular P pool, the use of [Ca2+]cyt may have to be restricted to avoid cytotoxic complexation of Ca2+ with limited P groups. Employing a range of genetically encoded Ca2+ reporters in Arabidopsis, P starvation but not nitrogen starvation was found to strongly dampen the root [Ca2+]cyt increases evoked by mechanical, salt, osmotic, and oxidative stress as well as by extracellular nucleotides. The strongly altered root [Ca2+]cyt response to extracellular nucleotides was shown to manifest itself during seedling development under chronic P deprivation, but could be reversed by P resupply. Fluorescent imaging elucidated that P-starved roots showed a normal [Ca2+]cyt response to extracellular nucleotides at the apex, but a strongly dampened [Ca2+]cyt response in distal parts of the root tip, correlating with high reactive oxygen species (ROS) levels induced by P starvation. Excluding iron, as well as P, rescued the altered [Ca2+]cyt response, and restored ROS levels to those seen under nutrient-replete conditions. P availability was not signalled through [Ca2+]cyt. In another part of this PhD project, a library of 77 putative Ca2+ channel mutants was compiled and screened for aberrant root hair growth under P starvation conditions. No mutant line showed aberrant root hair growth. These results indicate that P starvation strongly affects stress-induced [Ca2+]cyt modulations. The data generated in this thesis further understanding of how plants can integrate nutritional and environmental cues, adding another layer of complexity to the use of Ca2+ as a signal transducer.

Die Aktivierung der Speichelsekretion erfolgt in der innervierten Speicheldrüse der Schabe <i>Periplaneta americana</i> durch die biogenen Amine Dopamin (DA) und Serotonin (5-HT). Die Acini der Speicheldrüse sezernieren einen Primärspeichel, der in den Ausführgängen modifiziert wird. Die durch DA und 5-HT aktivierten Signalwege sowie die an der Elektrolyt- und Flüssigkeitssekretion bzw. Speichel-modifikation beteiligten Transportmechanismen sind weitgehend unbekannt.<br> Mikrofluorometrische Ca²⁺-, Na⁺- und pH-Messungen in Kombination mit pharmakologischen Experimenten, biochemische Messungen der Aktivitäten von Ionentransport-ATPasen sowie videomikroskopische Analysen zu transepithelialen Wasserbewegungen wurden in dieser Arbeit durchgeführt. Sie sollten Informationen über die an der Speichelbildung und -modifikation beteiligten Transportmechanismen und die Signalwege liefern, welche durch DA und/oder 5-HT aktiviert werden. <br><br> Wesentliche Ergebnisse dieser Arbeit waren:<br><br> <ul> <li>Messungen des intrazellulären pH (pH_i) in Gangzellen zeigten, dass isolierte Ausführgänge mit Acini bei Stimulierung mit DA und 5-HT stark ansäuerten. In isolierten Ausführgängen ohne Acini verursachte nur DA eine schwache Ansäuerung. Da nur die Ausführgänge dopaminerg innerviert sind, die Acini jedoch dopaminerg und serotonerg, zeigt dieses Ergebnis, dass die DA- und/oder 5-HT-induzierte Primärspeichelbildung die Ursache für die pHi-Änderungen in den Gangzellen ist. pH_i-Messungen in den Gangzellen geben also auch Hinweise auf Transportvorgänge in den Acini.</li> <li> Der Na⁺-K⁺-2Cl^--Symporter und der Cl^--HCO₃^--Antiporter, gekoppelt mit dem Na⁺ H⁺-Antiporter (NHE) waren an der NaCl-Aufnahme in die peripheren Zellen der Acini zur Bildung des NaCl-reichen Primärspeichels beteiligt. Die Aktivität dieser Transporter hing von der CO₂/HCO₃^--Verfügbarkeit ab und war Ca²⁺-abhängig.</li> <li>Die starke Ansäuerung in den Gangzellen hing nicht von der Aktivität der apikalen vakuolären Protonen-ATPase (V-H⁺-ATPase), aber von der Aktivität der basolateralen Na⁺-K⁺-ATPase ab, die anscheinend in den Ausführgängen die Speichelmodifikation energetisiert.</li> <li>In isolierten Ausführgängen mit Acini waren die V-H⁺-ATPase und Na⁺-abhängige Transporter (u. a. NHE) an der Erholung von einer DA-induzierten oder einer NH₄Cl-Vorpuls-induzierten Ansäuerung in den Gangzellen beteiligt. Bei der Regulation des pH_i in unstimulierten Gangzellen spielten diese Transporter keine Rolle.</li> <li>In isolierten Ausführgängen mit Acini induzierte DA in den Gangzellen einen Anstieg der [Na⁺]_i und, zeitlich verzögert, auch der [Ca²⁺]_i. Der [Na⁺]_i-Anstieg war von der Aktivität der Acini abhängig und erfolgte möglicherweise über apikale Na+-Kanäle. Der [Ca²⁺]_i-Anstieg war graduiert und tonisch. Der DA-induzierte [Na⁺]_i-Anstieg in den Gangzellen und deren Depolarisation führten dazu, dass der basolaterale Na⁺-Ca²⁺-Antiporter in den Ca²⁺-Influx-Modus umkehrte. Die daraus resultierende tonische [Ca²⁺]_i-Erhöhung könnte an der Regulation der Na⁺-Rückresorption beteiligt sein.</li> <li>Zum Nachweis transepithelialer Flüssigkeitsbewegungen in isolierten Ausführgängen wurde eine videomikroskopische Methode entwickelt. Isolierte Ausführgänge ohne Acini resorbierten im unstimulierten Zustand Flüssigkeit aus dem Ausführganglumen. Möglicherweise sezernieren die Acini auch im unstimulierten Zustand mit geringerer Rate einen Primärspeichel, der in den Ausführgängen resorbiert wird. Die Resorption war ATP-abhängig. Der ATP-verbrauchende Transportmechanismus konnte nicht identifiziert werden. Weder die Na⁺-K⁺-ATPase noch die V-H⁺-ATPase waren an der Resorption beteiligt.</li> </ul> <br> Diese Arbeit trug zur Kenntnis der komplexen Funktionsweise von Speicheldrüsen in Insekten bei und erweiterte das lückenhafte Wissen über die zellulären Wirkungen biogener Amine in Insekten. Zudem wurden in dieser Arbeit viele Parallelen zu Funktionsweisen der Speicheldrüsen in Vertebraten deutlich. / The acinar salivary glands in the cockroach <i>Periplaneta americana</i> are innervated by dopaminergic and serotonergic fibers and secrete a NaCl-rich primary saliva upon stimulation with the biogenic amines dopamine (DA) or serotonin (5-HT). The ducts downstream of the acini are thought to modify the primary saliva by Na⁺reabsorption and K⁺ secretion. The electrolyte and fluid transport processes activated by DA and 5-HT as well as the second messenger pathways mediating between the biogenic amine receptors and the effector transport mechanisms are poorly understood.In this sudy, microfluorometrical Ca²⁺, Na⁺ and pH measurements were performed in combination with pharmacological experiments. Furthermore, ATPase activity assays and microscopical analyses of transepithelial fluid transport were done. The aim of this work has been the characterisation of the DA-induced transport mechanisms in the cockroach salivary glands in order to improve our understanding of the cellular actions of biogenic amines in insects. <br><br> Intracellular pH measurements in duct cells of isolated small lobes of salivary glands consiting of several acini and ducts showed a strong intracellular acidification upon DA or 5-HT stimulation. On the other hand, only a small intracellular acidification could be recognised in isolated ducts without acini. The acini are innervated by dopaminergic and serotonergic fibers, whereas the ducts are innervated only by dopaminergic fibers. Thus, this result demonstrates, that the DA- or 5-HT-induced production of primary saliva in the acini causes the intracellular pH changes in the ducts. Consequently, intracellular pH measurements in ducts are also useful to characterise transport processes in the acini.<br><br> The Na⁺-K⁺-2Cl^- cotransport and/or the Cl^--HCO₃^- exchange combined with the Na⁺ H⁺ exchange (NHE) were responsible for the NaCl uptake at the basolateral membrane in the peripheral cells of the acini during production of primary saliva. The activity of these transporters was regulated by the CO₂/HCO₃^--availability and was Ca²⁺-dependent. The activity of the basolateral Na⁺-K⁺-ATPase, but not of the apical vacuolar-type proton pump (V-H⁺-ATPase) in the duct cells was necessary for the strong intracellular acidification in the ducts with acini. Thus, the Na⁺-K⁺-ATPase seems to energise the saliva modification in the ducts. In ducts with acini, the V-H⁺-ATPase and Na⁺-dependent transporters (e.g. NHE) were responsible for the pH-recovery after a DA- or NH₄Cl-induced intracellular acidification in the duct cells. In the regulation of the intracellular resting pH these transporters played a minor role. In addition, DA induced an increase in the intracellular Na⁺ concentration, followed by an increase in the intracellular Ca²⁺ concentration in duct cells with acini, but never in duct cells without acini. The Na⁺ elevation was probably the result of the activity of apical Na⁺ channels. The DA-induced Na⁺ elevation and a depolarisation of the basolateral membrane of the duct cells reversed a Na⁺-Ca²⁺ exchange activity into the reverse mode causing a graded Ca²⁺ elevation in duct cells. The Ca²⁺ elevation is probably involved in the regulation of the Na⁺ reabsorption during saliva modification. Transepithelial fluid transport in isolated ducts was detected with a fluorescent microscopical method. Already unstimulated isolated ducts reabsorbed fluid from the duct lumen to the bath side. Perhaps unstimulated acini possess a basic secretion rate and this primary saliva is than reabsorbed in the ducts. The fluid reabsorption was ATP-dependent, but the ATP-consuming transport mechanism could not be identified. Neither the basolateral Na⁺-K⁺-ATPase, nor the apical V-H⁺-ATPase were involved in fluid reabsorption. This work extends our knowledge about the complex function of insect salivary glands and about the cellular action of biogenic amines in insects. Additionally, it indicates lots of similarities between the functions of salivary glands in vertebrates and invertebrates.

La méthylation de l'ADN est une des marques épigénétiques majeures qui intervient dans la régulation de processus physiologiques importants. Par ailleurs, elle a été reconnue comme une cible majeure pour lutter contre le cancer car son dysfonctionnement est impliqué dans le développement de pathologies comme les cancers. Une meilleure compréhension de ce mécanisme permettrait aux chimistes médicinaux de développer de nouveaux inhibiteurs plus spécifiques. Actuellement, un grand nombre d'hypothèse ont été avancées concernant la méthylation de l'ADN et méritent d'être confirmées ou clarifiées. Dans ce contexte, nous avons décidé de développer de nouveaux analogues nucléosidiques fluorescents et ratiométriques présentant un motif 3-hydroxychromones, afin d'étudier la dynamique et le mécanisme de méthylation de la cytosine impliquant UHRF1 et DNMT1. Nous avons tout d'abord décidé de nous focaliser sur la première étape du mécanisme, la reconnaissance du duplexe hémi-méthylé par le domaine SRA de UHRF1. Deux stratégies de marquage de l'ADN ont ainsi été explorées. La première a consisté à remplacer une base azotée naturelle par le fluorophore, afin de sonder l'intérieur du duplexe. Ce biocapteur a ainsi pu être utilisé afin de détecter l'approche du domaine SRA de UHRF1 ainsi que le basculement de la cytosine méthylée dans son site de reconnaissance. La deuxième approche repose sur la modification d'une nucléobase naturelle sur laquelle la sonde ratiométrique est attachée à l'aide d'un bras espaceur insaturé rigide, lui permettant ainsi de sonder les interactions au niveau du grand sillon de l'ADN. / DNA methylation is one the major epigenetic modification and plays an important role in the regulation of major processes. DNA methylation was recently recognized as one of the major target to inhibit and to fight cancer. A deeper insight of the DNA methylation mechanism should help medicinal chemists in the design and research of more specific inhibitors of DNA methylation. Currently, miscellaneous hypotheses are advanced about DNA methylation mechanism and deserve to be confirmed or clarified. In this context, we decided to develop new ratiometric fluorescent nucleoside analogs, incorporating 3-hydroxychromone moiety, to study the dynamics and mechanism of cytosine methylation involving UHRF1 and DNMT1. We firts decided to focus on the first step of the mechanism, the recognition of the hemi-methylated duplex by the SRA domain of UHRF1. Two diferent strategies of labeling DNA were explored. In the firts one, a natural nucleobase was substituted by the fluorescent dye in order to probe inside the duplex. This biosensor allows to detect and monitor the binding of the SRA domain and flipping of the 5-methylcytosine from the duplex into its recognizing site. The second approach is based on a modification of a natural nucleobase on which, through a rigid unsaturated linker, the ratiometric probe was incorporated in order to sense the interaction in the major groove.

The diploma thesis evaluates the economic situation of the company Niveko s.r.o. in between years 2011 to 2018. The theoretical part describes financial indicators, time series, regression and correlation analysis. The analytical part contains calculations of financial indicators, some of which are selected for statistical analysis, which is used to determine the expected development of indicators in the next two years or to reveal the dependence between selected indicators. The last part contains suggestions for improving the current economic situation of the company.

The aim of my thesis is the financial assessment of selected construction company through financial analysis. The theoretical part includes characterization of construction company, financial statements and a description of the financial analysis and its methods. The practical part evaluates the economical situation of company through financial analysis. The financial analysis is prepared for methods of analysis of summary indicators, horizontal and vertical analysis and analysis of ratiometric indicators.

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

In der vorliegenden Dissertation konnten erfolgreich neben der Entwicklung und Optimierung neuer Methoden in der Kettenverlängerung von Aldosen und Ketosen auch neue Farbstoffe zur Untersuchung von Protonierungs- und Wasserdynamiken designt und synthetisiert werden. Die Erweiterung des Verständnisses im Zusammenspiel von Aminosäuren und Kohlenhydraten hat einen großen Einfluss in der wissenschaftlichen Gesellschaft der Kohlenhydratchemie. Die Kontrolle im Aufbau von Stereotetraden und -pentaden führt zu einer Ausweitung der bekannten Möglichkeiten in der stereoselektiven Synthese von Naturstoffen und Biomimetika. Außerdem konnten durch eine einfache Methode die C-Glykoside seltener Kohlenhydrate dargestellt werden. Einbau von Aminosäuren in Kohlenhydratstrukturen konnten in hohen Stereoselektivitäten und der Möglichkeit der Stereomanipulation durch Wahl des Isocyanides erreicht werden. Die Synthese des zellgängigen Farbstoffes PAc-SNARF, sowie des Biolinkers IA-SNARF ermöglicht eine bildgebenden ratiometrischen pH-Untersuchungen in Zellen und auf Proteinoberflächen. Die Verallgemeinerung der Synthese der Farbstoff-Precursor für die SNARF-Derivate über eine Friedel-Crafts-Acylierung erlaubt eine kostengünstige Darstellung einer großen Bandbreite von Farbstoffen. Mithilfe der neu synthetisierten sterisch anspruchsvollen N-Methyl-6-oxychinoliniumbetain-Derivate verbunden mit der Fluoreszenzaufkonvertierungs-Spektroskopie konnte eine Verlangsamung von Wasser an hydrophoben Oberflächen bewiesen werden. Die gesammelten Ergebnisse und Erkenntnisse in diesen verschiedenen Themengebieten werden in Zukunft einen großen Einfluss in der Wissenschaftswelt haben. / In the presented dissertation new methods in the chain elongation of carbohydrates could be established and optimized. Besides that, new probes for the investigation of protonation and water dynamics could be designed and synthesized. The extension of comprehension in the interaction between amino acids and carbohydrates through hydrogen bonds has a great impact in the scientific community of carbohydrate research. The stereochemical control in the construction of stereotetrads and –pentads leads to a considerable extension of known methods in the synthesis of natural compounds and biomimetics. Additionally the C-glycosides of rare carbohydrates could be synthesized through simple methods. Installation of amino acids into carbohydrate structures could be achieved with very high stereoselectivity and the potential of manipulating the stereochemical course through the choice of different isocyanides. The synthesis of the cell permeable PAc-SNARF and the cysteine-bioapplicable IA-SNARF allow the ratiometric pH-imaging of cells and protein surfaces. The generalization of the synthesis of dye-precursors for SNARF-derivatives through friedel-crafts-acylation allow an inexpensive approach in synthesizing a broad spectrum of dyes. Through deployment of newly developed sterical demanding N-Methyl-6-oxyquinolinium betaine-derivates together with the fluorescence upconversion spectroscopy a deceleration of water reorientation near hydrophobic surfaces could be proven. The here summarized results and insights in the different topics will have a considerable influence in academic sciences.

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No. of bitstreams: 2 GABRIEL_Gabriele_2017.pdf: 4796378 bytes, checksum: b47c6fae3bd085c69ed8a81c74a9be4d (MD5) GABRIEL_Gabriele_carta.pdf: 557241 bytes, checksum: fbed02fe69c0cab6190201e18b304f40 (MD5) Previous issue date: 2017-12-01 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Bioluminescence, the emission of visible light by living organisms is widely used in biosensors. Firefly luciferases and genes are among the most used reporter gene in bioluminescent biosensors. Firefly luciferases are pH-sensitive, exhibiting a red shifted bioluminescence spectra on the presence of metals, high temperatures and acidic pH, being this last property considered unusual for the most analytical applications. Nowadays most luminescent biosensors to metals and pH are fluorescent, and few of them are ratiometric based on spectral changes. Bioluminescent biosensors despite been less common, have same advantages as low background and does not need UV light irradiation, bring no photodamage to the cell. The aim of this project was: (I) study the applicability of the use of Macrolampis sp2 firefly luciferase and other pH-sensitive luciferases as spectral intracellular ratiometric pH biosensor; (II) apply these pH ratiometric biosensors in mammalian cells to investigate its physiology in real time and (III) developing, by engineering the pH sensor region of Macrolampis sp2 firefly luciferase, a ratiometric biosensor specific to metals. We obtained a relation between the pH and the ratio of the bioluminescence at green and red region of the spectra, allowing estimate ratiometrically the intracellular pH of bacteria and mammalian cells. Besides that, we confirmed its use to cellular image of pH and observed that occurs an alkalinization of the cytosol and nucleus during cell division and apoptosis. We demonstrate the applicability of firefly luciferases, in special the luciferase of Macrolampis sp2, as a ratiometric biosensor to metals and intracellular pH. The existence of a linear relationship between the concentrations of metals like cadmium, mercury and zinc, and the ratio of the bioluminescence at green and red region of the spectra, allowed also estimate ratiometrically, for the first time, concentrations of metals less than 100 µM, enabling the use of firefly luciferases as bioavailability indicator to toxic and potential toxic metals. / Bioluminescência, a emissão de luz fria e visível por organismos vivos é amplamente utilizada em biossensores. Luciferases de vagalumes e seus genes estão entre os genes repórteres mais utilizados em biossensores bioluminescentes. Luciferases de vagalumes são sensíveis ao pH, apresentando um deslocamento do espectro de bioluminescência para o vermelho na presença de metais, em temperaturas elevadas e pH ácido, sendo esta última propriedade considerada sem utilidade para a maioria das aplicações analíticas. Atualmente a maioria dos biossensores luminescentes para metais e pH são fluorescentes, poucos deles são raciométricos baseados nas mudanças espectrais. Biossensores bioluminescentes apesar de serem menos comuns, possuem algumas vantagens como baixo background e não necessitam de irradiação com luz UV, não causando fotodanos às células. Os objetivos desse projeto foram: (I) investigar a aplicabilidade de uso da luciferase do vagalume Macrolampis sp2 e outras luciferases pH-sensitivas como biossensor espectral raciométrico intracelular de pH; (II) aplicar esses biossensores raciométricos de pH em células de mamíferos para investigar sua fisiologia em tempo real e (III) desenvolver, por engenharia da região sensora de pH da luciferase de Macrolampis sp2, um biossensor raciométrico específico para metais. Obtivemos uma relação entre o pH e a razão da bioluminescência nas regiões do verde e do vermelho do espectro, permitindo estimar raciometricamente o pH intracelular de bactérias e células de mamíferos. Além disso, confirmamos seu uso para imagem celular de pH, e observamos que ocorre uma alcalinização do citosol e núcleo durante os processos de divisão celular e apoptose. Demonstramos a aplicabilidade das luciferases de vagalumes, em especial a luciferase de Macrolampis sp2, como biossensor raciométrico para metais e pH intracelular. A existência de uma relação linear entre a concentração de metais como cádmio, mercúrio e zinco, e a razão da bioluminescência nas regiões do verde e do vermelho do espectro, permitiu também estimar raciometricamente pela primeira vez concentrações de metais menores que 100 µM, possibilitando o uso de luciferase de vagalumes como indicador de biodisponibilidade de metais tóxicos e potencialmente tóxicos. / FAPESP: 2014/04477-9 / FAPESP: 2015/22603-4 / FAPESP: 2016/15946-5

The development of shape in multicellular organisms has intrigued human minds for millenia. Empowered by modern genetic techniques, molecular biologists are now striving to not only dissect developmental processes, but to exploit their modularity for the design of custom living systems used in bioproduction, remediation, and regenerative medicine. Currently, our capacity to harness this potential is fundamentally limited by a lack of spatiotemporal control over gene expression in multicellular systems. While several synthetic genetic circuits for control of multicellular patterning have been reported, hierarchical induction of gene expression domains has received little attention from synthetic biologists, despite its fundamental role in biological self-organization. In this thesis, I introduce the first synthetic genetic system implementing population-based AND logic for programmed hierarchical patterning of bacterial populations of Escherichia coli, and address fundamental prerequisites for implementation of an analogous genetic circuit into the emergent multicellular plant model Marchantia polymorpha. In both model systems, I explore the use of bacteriophage T7 RNA polymerase as a gene expression engine to control synthetic patterning across populations of cells. In E. coli, I developed a ratiometric assay of bacteriophage T7 RNA polymerase activity, which I used to systematically characterize different intact and split enzyme variants. I utilized the best-performing variant to build a three-color patterning system responsive to two different homoserine lactones. I validated the AND gate-like behavior of this system both in cell suspension and in surface culture. Then, I used the synthetic circuit in a membrane-based spatial assay to demonstrate programmed hierarchical patterning of gene expression across bacterial populations. To prepare the adaption of bacteriophage T7 RNA polymerase-driven synthetic patterning from the prokaryote E. coli to the eukaryote M. polymorpha, I developed a toolbox of genetic elements for spatial gene expression control in the liverwort: I analyzed codon usage across the transcriptome of M. polymorpha, and used insights gained to design codon-optimized fluorescent reporters successfully expressed from its nuclear and chloroplast genomes. For targeting of bacteriophage T7 RNA polymerase to these cellular compartments, I functionally validated nuclear localization signals and chloroplast transit peptides. For spatiotemporal control of bacteriophage T7 RNA polymerase in M. polymorpha, I characterized spatially restricted and inducible promoters. For facilitated posttranscriptional processing of target transcripts, I functionally validated viral enhancer sequences in M. polymorpha. Taking advantage of this genetic toolbox, I introduced inducible nuclear-targeted bacteriophage T7 RNA polymerase into M. polymorpha. I showed implementation of the bacteriophage T7 RNA polymerase/PT7 expression system accompanied by hypermethylation of its target nuclear transgene. My observations suggest operation of efficient epigenetic gene silencing in M. polymorpha, and guide future efforts in chassis engineering of this multicellular plant model. Furthermore, my results encourage utilization of spatiotemporally controlled bacteriophage T7 RNA polymerase as a targeted silencing system for functional genomic studies and morphogenetic engineering in the liverwort. Taken together, the work presented enhances our capacity for spatiotemporal gene expression control in bacterial populations and plants, facilitating future efforts in synthetic morphogenesis for applications in synthetic biology and metabolic engineering.