Dissertations / Theses on the topic 'Luciferasi'

Luciferase catalyzes the emission of blue-green light and is the central feature of bacterial bioluminescence. The three-dimensional structure of the bacterial luciferase apo-enzyme determined by X-ray crystallography has revealed the detailed landscape of the enzyme active site, however, the absence of a structure with bound substrate has impeded the understanding of the enzyme mechanism by which luciferase interacts with substrates and catalyzes their conversion into light emission. This thesis describes three research projects that focus on the molecular conformation of flavin in the active site of bacterial luciferase. Based on available structure-activity data as guidance, the first project deduces the binding conformation of the luciferase bound flavin by computer modeling. The second research project investigates the binding microenvironment of flavin, and assigns specific functions to the structural modules (amino acid residues), which coordinate flavin in the proposed model. The third project verifies the validity of the proposed model with mutational analysis of the binding site residues, and points out the possibility of altering the visible emission color of bacterial bioluminescence by redesigning luciferase. The last chapter concludes the thesis with the discussion of the structural mechanism of luciferase catalysis, and the perspectives for the engineering of luciferase variants that emit different light colors.

Esta tese descreve como é possível obter emissão de luz in vitro enzimaticamente, a partir de extratos quente e frio de diferentes espécies de fungos bioluminescentes, o que indica também um mecanismo comum de bioluminescência em todos esses organismos. Dados cinéticos sugerem um mecanismo enzimático em duas etapas e corroboram a hipótese enzimática de Airth e Foerster, da década de 1960. Finalmente, utilizando-se extratos quente e frio foi possível também isolar a luciferina fúngica e obter sua massa molecular (298,1837 m/z). Essa substância isolada emite luz enzimaticamente in vitro, sendo que a sobreposição do espectro de emissão e do espectro de bioluminescência do fungo confirma que essa substância é a luciferina fúngica.
This thesis describes how in vitro light emission can be enzymatically obtained from the hot and cold extracts assay using different species of fungi, which also indicates a common mechanism of light emission for all these organisms. Kinetic data suggest a consecutive two-step mechanism and corroborate the 1960\'s enzymatic proposal of Airth and Foerster. Finally, using hot and cold extracts assay we were also able to purify and to determine the molecular weight of the fungal luciferin (298.1837 m/z). The isolated substance emits light enzymatically in vitro, whose light emission spectrum matches with the fungal bioluminescence one thus confirming that the substance is the fungal luciferin

Les macrolides forment un groupe homogène d'agents antibactériens produits par des Streptomyces et d'autres actinomycètes. Ils inhibent la synthèse protéique des bactéries par liaison à la sous-unité ribosomique 50S, ce qui empêche son assemblage ou son fonctionnement. La première partie de ce manuscrit porte sur l'effet des macrolides sur la fidélité de traduction. Nous avons utilisé comme système rapporteur la luciférase de Vibrio harveyi avec un codon stop introduit au début du gène luxB pour mesurer in vivo la suppression de ce codon stop. L'érythromycine augmente la trans-lecture du codon stop UAG et diminue donc la fidélité de traduction, en accord avec l'hypothèse selon laquelle les macrolides agissent en début d'élongation. Cet effet a été confirmé par l'étude du taux global d'erreur en utilisant l'électrophorèse 2-D des protéines. La deuxième partie porte sur la résistance naturelle aux macrolides chez les mycobactéries du complexe M. Tuberculosis (CMT), résistance généralement attribuée à la structure spécifique de la paroi des mycobactéries. Nous avons identifié un gène codant une méthyltransférase de type Erm, modifiant les ribosomes et conférant la résistance aux macrolides. Ce gène, appelé ermMT, est conservé chez tous les membres du CMT mais est affecté par une délétion dans certaines souches vaccinales de M. Bovis BCG comme BCG Pasteur. Cette dernière souche est sensible aux macrolides, alors que les autres membres du CMT sont résistants. L'expression de ermMT dans des mycobactéries sensibles aux macrolides confère la résistance. La comparaison des niveaux de résistance et de l'affinité pour l'érythromycine des ribosomes de souches exprimant ermMT ou d'autres gènes erm indique que ermMT confère une résistance de type I aux macrolides, lincosamides et streptogramines, correspondant à la mono-méthylation de A2058 dans l'ARN 23S. Nos résultats montrent que ermMT joue un rôle majeur dans la résistance naturelle aux macrolides chez les mycobactéries du CMT
Macrolide antibiotics constitute a homogenous group of antibacterial agents produced by Streptomyces or related Actinobacteria. They inhibit protein synthesis in bacteria by binding to the 50S ribosomal subunit, preventing its assembly or inhibiting its function. In the first part of this thesis the effect of macrolides on translation accuracy was studied. We have used the reporter system based on Vibrio harveyi luciferase with a stop codon inserted in the proximal part of the luxB gene for the in vivo measurement of the nonsense codon readthrough. Erythromycin stimulated the leadthrough of the UAG stop codon and thus the decrease of the translation accuracy. This is in agreement with the hypothesis that macrolides influence the early stages of elongation process. The misreading effect of macrolides was confirmed by the study of global error frequencies using the 2-D gel electrophoresis of proteins. The second part deals with the intrinsic macrolide resistance of the Mycobacterium tuberculosis complex (MTC), generally attributed to the low permeability of the mycobacterial cell wall. However we have shown that a gene, whose product confers macrolide resistance by ribosome modification, was present in all members of the MTC. It was named ermMT (erm. 37). Part of the ermMT is deleted in some vaccinal strains of Mycobacterium bovis BCG, such as the Pasteur strain. The Pasteur strain was susceptible to macrolides, whereas MTC species were resistant to them. The expression of ermMT in the macrolide-sensitive Mycobacterium strains conferred macrolide resistance. Comparison of the resistance patterns and ribosomal affinity for erythromycin of Mycobacterium host strains expressing ermMT or other erm genes indicates that ermMT confers a type I resistance to macrolides, lincosamides and streptogramins, coiresponding to the mono-methylation of A2058 in 23S rRNA. Our results indicate that ermMT plays a major role in the intrinsic macrolide resistance of members of the MTC

Pyrosequencing is a new DNA sequencing method relying on thesequencing-by-synthesis principle and bioluminometric detectionof nucleotide incorporation events. The objective of thisthesis was improvement of the Pyrosequencing method byincreasing the thermal stability of firefly luciferase, and byintroducing an alternative DNA polymerase and a new nucleotideanalog. Furthermore, the development of a new bioluminescentassay is described for the detection of inorganicpyrophosphatase activity.

The wild-type North American firefly(Photinus pyralis)luciferase is a heat-sensitiveenzyme, the catalytic activity of which is rapidly lost attemperatures over 30°C. Two strategies for increasing thethermostability of the enzyme are presented and discussed. Inthe first strategy, the solution thermodynamics of the systemis affected by osmolytes in such a way that heat-mediatedinactivation of the enzyme is prevented. In the secondstrategy, the enzyme is thermostabilized by mutagenesis. Bothstabilizing strategies can be utilized to allow bioluminometricassays to be performed at higher temperatures. For instance,both DNA polymerase and ATP sulfurylase activity could beanalyzed at 37°C.

The osmolyte strategy was successfully employed forincreasing the reaction temperature for the Pyrosequencingmethod. By increasing the reaction temperature to 37°Cunspecific signals from primer-dimers and 3’-end loopswere reduced. Furthermore, sequencing of a challenging templateat 37°C, which previously yielded poor, non-interpretablesequence signals at lower temperatures was now possible.

Introduction of a new adenosine nucleotide analog,7-deaza-2’-deoxyadenosine-5’-triphosphate (c⁷dATP) reduced the inhibitory effect on apyraseobserved with the currently used analog,2’-deoxyadenosine-5’-O-(1-thiotriphosphate)(dATPαS).

Sequencing of homopolymeric T-regions has previously beendifficult with the exonuclease-deficient form of the DNApolymerase I large (Klenow) fragment. By using the DNApolymerase from bacteriophage T7, known as Sequenase, templateswith homopolymeric T-regions were successfully sequenced.Furthermore, it was found that the strand displacement activityfor both polymerases was strongly assisted if the displacedstrand had a 5’-overhang. In contrast, the stranddisplacement activity for both polymerases was inhibitedwithout an overhang, resulting in reduced sequencingperformance in double stranded regions.

A firefly bioluminescent assay for the real-time detectionof inorganic pyrophosphatase in the hydrolytic direction wasalso developed. The assay is versatile and has a linearresponse in the range between 8 and 500 mU.

Key words:bioluminescence, osmolytes, glycine betaine,thermostability, firefly luciferase, inorganic pyrophosphatase,inorganic pyrophosphate, Pyrosequencing technology, secondaryDNA-structures, Sequenase, Klenow-polymerase, reaction rates,temperature, c⁷dATP, dATPαS.

A leishmaniose é causada por protozoários do gênero Leishmania e no Brasil, as principais espécies causadoras da leishmaniose cutânea são Leishmania (V.) braziliensis e Leishmania (L.) amazonenses. O tratamento da leishmaniose apresenta diversas dificuldades, portanto é fundamental a descoberta de novos fármacos ativos, podendo ser detectada em células cultivadas in vitro e também em animais íntegros, através da técnica de bioimageamento. Neste trabalho, propusemo-nos a produzir linhagens de L. (V.) braziliensis e L. (L) amazonenses expressoras de luciferase e caracterizar o comportamento das linhagens mutantes em testes de sensibilidade a fármacos e de infecção in vitro e in vivo. Foi confirmada a emissão de luz pelas linhagens mutantes das duas espécies de Leishmania, em promastigotas e amastigotas. O comportamento das linhagens mutantes obtidas em relação a curvas de crescimento, sensibilidade aos fármacos tamoxifeno e anfoterina B em promastigotas, perfil de infectividade e sobrevivência em macrófagos e sensibilidade de amastigotas à anfotericina B foi comparado ao comportamento das linhagens parentais, não sendo observadas diferenças significativas. Camundongos BALB/c infectados com a linhagem expressora de luciferase de L. (L.) amazonenses desenvolveram lesões comparáveis aos animais infectados com a cepa selvagem, sendo possível quantificar a carga parasitária nesses animais por bioimageamento. Os resultados obtidos neste trabalho indicam que os parasitas mutantes expressores de luciferase obtidos podem ser utilizados em testes de sensibilidade a fármacos tanto in vitro como in vivo, representando um avanço metodológico nessa área de pesquisa.
Leishmaniasis is caused by protozoan parasites in Brazil, the main causative species of cutaneous leishmaniasis are Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonenses. The treatment of leishmaniasis presents several difficulties, and the discovery of new active drugs for the treatment of leishmaniasis is therefore fundamental. The enzyme luciferase is a reporter widely used in screening tests for new drugs. This enzyme catalyzes the oxidation of luciferase in the presence of ATP emitting light that can be detected in cultured cells in vitro as well as in intact animals, using the technique of bioimaging. In this work, we sought to produce strains of L. (V.) braziliensis and L. (L) amazonenses expressing luciferase and characterize the behavior of these mutant strains in drug susceptibility tests and in in vitro and in vivo infections. Production of light was detected in mutants of both species, in all life cycle stages. Mutant strains were compared to their corresponding parental lines as to their growth pattern, infectivity and survival profile in macrophages and sensitivity to amphotericin B and tamoxifen. No significant differences were observed for these parameters. BALB/c mice infected with the luciferase expressing line of L. (L.) amazonenses developed lesions comparable to those in animals infected with the wild-type strain. The parasite load in these animals was quantified through bioimaging. The results obtained of this study indicate that the mutant parasites expressing luciferase can be used for drug susceptibility testing in vitro and in vivo, representing a methodological advance in this area of research.

Firefly luciferase is ubiquitously used as a genetic reporter for the non-invasive bioluminescence imaging of small animal models. This widespread use of Firefly luciferase in vivo has been facilitated by genetic engineering producing mutants which are extremely stable at physiological conditions. In addition, the red-shifting of bioluminescence has resulted in the enhanced penetration of light emission through biological tissue. However, the use of bioluminescence in vivo is still largely limited to the tracking of single events within a model. This is due to the differential attenuation of light < 600nm, making the spectral unmixing of bioluminescent signals extremely challenging. Consequently, there is a real need to move bioluminescence into the near-infrared for dual-colour imaging. As we seem to have reached the limits of mutational based red-shifting, research has more recently focused upon chemical modification of the D-luciferin substrate. But any modification of the DLuciferin substrate is inevitably going to require subsequent mutagenesis of Firefly luciferase to optimise the light emitting reaction. The first part of this project describes the development and validation of a high throughput screening platform for bioluminescent proteins, to advance the identification of mutants with enhanced characteristics. Focus then turns to the use of genetically engineered Firefly luciferase colour mutants for in vivo bioluminescence imaging. Small animal tumour models, representing increasing tissue depth, were engrafted with Firefly luciferase colour mutants to explore the feasibility of dual colour imaging and establish the true benefit of red-shifting bioluminescence. Finally, bioluminescence in the near infra-red is used for dual bioluminescence imaging, tracking two tumour populations in a B-cell lymphoma mouse model through the spectral unmixing of Firefly luciferase colour mutants with the novel substrate infra-luciferin.

This licentiate thesis presents computational chemistry studies on photochemical properties of phytochrome and firefly luciferase chromophores. Phytochromes are bilin-containing proteins that based on the ambient light environment regulate a number of physiological and developmental processes in bacteria, cyanobacteria, fungi and plants. From the viewpoint of computational modeling, however, only a few studies have been devoted to these systems. In this thesis, two systematic studies comparing calculated and experimental UV-vis spectra of bilin chromophores in protein and solution environments are presented. The rst study focuses on how hybrid quantum mechanics/molecular mechanics methods are best applied to calculate absorption spectra of a bacteriophytochrome. The second study, in turn, investigates the performance of a number of quantum chemical methods in calculating absorption and emission spectra of sterically locked bilin chromophores. Firefly luciferase catalyzes a chemical reaction in which the electronically excited oxyluciferin is formed and subsequently emits light. Depending on the conditions, oxyluciferin can exist in a number of dierent chemical forms. To date, there is no consensus regarding which of these that most signicantly contributes to the light emission. In this thesis, the most probable form of the light emitter is predicted by calculating excited-state pKE and pKa values, in aqueous solution, of the various equilibrium reactions relevant for the oxyluciferin system.

The bioluminescence reaction is catalysed by firefly luciferase, converting the substrates D-luciferin, ATP and molecular oxygen with Mg2+ to produce light and this reaction has had wide ranging implications within a number of fields from industry to academia. The discovery of luciferase has been revolutionary in the real time in vivo study of cells given that it requires no energy for excitation, delivering a high signal to background ratio providing a highly sensitive assay. This protein, to date, has been utilised in molecular cell biology, cellular imaging, microbiology and numerous other fields. The extensive application of this protein has paved the way for the generation of toolbox of variants with altered properties. Protein engineering involving substitution mutations made within Photinus pyralis (Ppy) FLuc has led to the discovery of a number of novel variants however there is a bank of growing evidence displaying the power of deletions as an alternative for the development of proteins with altered properties since deletions can sample structural diversity not accessible to substitutions alone. A novel mutagenic strategy was implemented to incorporate single amino acid deletions within thermostable firefly luciferase (x11FLuc) targeting loop structures (M1-G10, L172- T191, T352-F368, D375-R387, D520-L526, K543-L550). Of 43 deletion mutants obtained, 41 retained bioluminescent activity and other characteristics such as resistance to thermal inactivation. Surprisingly, only 2 variants, ΔV365 and ΔV366, exhibited a complete loss of activity showing that the luciferase protein is largely tolerant to single amino acid deletions. In order to identify useful mutants in the extensive library, a 96-well format luminometric cell lysate assay was developed which indicated the effect of deletions was largely region specific, for example, N- terminal deletions did not alter the activity of x11FLuc, whilst deletions within L172- T191, D375-R387, D520-L526 and the C- terminal loop reduced overall activity. On the other hand, deletions within T352-F368 enhanced overall bioluminescent activity and remarkably exhibited other important characteristics such as increases in specific activity and a reduced KM for luciferin. Therefore, a novel motif (omega loop) was identified as important for FLuc activity after full characterization of mutants. Characterisation of the deletion mutants originating from the omega loop (T352-F368), ΔP359 and ΔG360 both presented a reduced KM for luciferin, whilst ΔA361, ΔV362, ΔG363 presented an increase in KM towards ATP as compared to x11FLuc. Thus, deletions in the omega loop, in the main, improved activity and altered reaction kinetics, in particular ΔG363 retained 53% of initial activity after 250s. As such, it is considered that the field of protein engineering should not only overlook the utility of single amino acid deletions, since such mutagenic strategies may sample structural space not achieved by substitutions alone and mutations within less popularized secondary structures such as omega loops are can act as a useful tool in the improvement of proteins.

Breast cancer is the most common cancer in the UK and approximately 1 in 8 women will be affected by the disease. Estrogen regulates breast cancer growth through the action of the estrogen receptors ERα and ERβ. Antiestrogens, in particular tamoxifen, have contributed greatly to the reduction in breast cancer mortality. Tamoxifen is a tissue selective antiestrogen; it is antiestrogenic in the breast but estrogenic in other tissues, thereby enabling it to promote the beneficial effects of estrogen, such as maintaining bone density. However, like estrogen, tamoxifen also promotes endometrial cancer, so there is an impetus for the development of novel tissue selective ERα ligands. Regulation of gene expression by the estrogen receptors requires the ligand-regulated recruitment of transcription coregulator proteins. In breast cancer ERα-coactivator interactions are associated with tumour progression while ERα-corepressor interactions are associated with receptor antagonism and a therapeutic block of ERα signalling. This thesis details the development of a luciferase fragment complementation assay to image the interaction of ERα with the coactivator AIB1 and corepressor SMRT. It is hoped that elucidation of these interactions will enable a greater appreciation of the tissue selective actions of ERα ligands and aid in the screening of novel ERα antagonists. By means of complimentary luciferase fragment fusion proteins, it is shown that ligand dependent ERα-coregulator interaction can be imaged in vitro and in vivo. ERα and AIB1 luciferase fusion proteins indicate an E2 induced increase in luciferase fragment complementation which is modulated by antiestrogens. The complementation observed correlates with ERα transcriptional activity and the specificity has been further validated by ERα fusion protein mutants. Consistent with the notion that the ERα-SMRT interaction is characteristic of ERα antagonism, ERα and SMRT fusion proteins show increased luciferase fragment complementation with antiestrogens compared with estrogen.

Split-protein reassembly methods allow for the detection of a variety of macromolecular interactions in vitro, in cellulo and in vivo. Spilt-protein reporters, such as split-firefly luciferase (Fluc), depend upon the conditional reassembly of the genetically fragmented enzyme. The two fragments of Fluc conditionally reassemble and regain activity when protein domains appended to the N-terminal and C-terminal Fluc fragments interact. Our laboratory has previously reported the use of the split-Fluc system in the detection of protein-protein, protein-nucleic acid, and protein-small molecule interactions. Herein, I describe further developments in the split-Fluc system as applied to the specific detection of DNA, DNA modifications, and small molecule kinase inhibitors both in vitro and in cellulo. We have previously developed one of the first direct methods to detect any double stranded DNA sequence by attaching DNA binding domains, specifically designed Cys₂-His₂ zinc finger domains, to split-Fluc fragments. We previously discovered that designed Cys₂-His₂ zinc fingers were not specific for targeting a variety of promoter sequences. In order to improve DNA detection, I have designed and tested split-Fluc sensors against several relevant DNA targets using both designed zinc fingers and transcription activator-like effectors (TALEs). The results demonstrate that TALEs allow for significantly higher specificity than zinc fingers and provide a significant improvement for designing sequence specific split-Fluc systems for a variety of applications such as the detection of DNA methylation. The methylation at cytosine-guanine (CpG) dinucleotides in DNA is associated with transcriptional repression in normal cells. Aberrant changes in the mCpG levels on the promoter regions of tumor suppressor genes have been linked to cancer. We have previously shown that a sequence-specific mCpG split-Fluc sensor can be designed by attaching one fragment of the split-Fluc to a zinc finger and another fragment to the methyl-binding domain (MBD) protein, which selectively binds mCpGs. In the long term we wanted to improve these sensors and focused upon understanding the details of MBD binding to mCpG sites. I have completed a complete alanine-scan of the MBD domain using our split-Fluc systems that allows for determining how each residue in MBD1 contributes to mCpG DNA binding. The results showed that numerous residues being necessary for mCpG recognition that have not been previously identified, especially residues that likely contribute to an essential hydrophobic core. These alanine scanning results along with the improvements in DNA recognition with TALEs will potentially allow for the design and selection of more selective mCpG sensors. In a further area related to DNA modifications, in this case DNA damage, we have developed split-Fluc sensors for detecting the post-translational modification on proteins, poly(ADP-ribosyl)ation utilizing PAR-binding zinc fingers of the protein APLF. I have designed and tested a variety of PAR binding sensors and also demonstrated the utility of these sensors for measuring PAR in mammalian cells upon induction of DNA damage. In a separate area, our laboratory has been interested in developing methods for understanding the selectivity of small molecule kinase inhibitors using split-Fluc methods. Protein kinases catalyze the transfer of the ɣ-phosphate group from ATP to an acceptor protein and are involved in almost all signal transduction events. The deregulation of protein kinases is associated with many diseases, particularly cancer, and there has been tremendous interest in developing kinase inhibitors for therapeutic use. One of the main challenges is the ability to understand inhibitor selectivity as the >500 protein kinases in human possess very similar structures especially at the ATP-binding site targeted by small molecules. Our laboratory has previously reported an in vitro three-hybrid screen for protein kinase inhibitors, where one split-Fluc fragment is attached to a protein kinase, and the other is attached to the peptide Fos. A chemical inducer of dimerization (CID), composed of a peptide Jun, which dimerizes with Fos, as well as the ATP-binding kinase ligand, induces the formation of a three-hybrid complex that reassembles the split-Fluc. Small molecule inhibitors can be identified by their ability to prevent the formation of the three-hybrid system, resulting in reduced luciferase activity. There are few direct methods to interrogate small molecule binding in mammalian cells. In order to interrogate inhibitors in cells, we have developed a cell-permeable CID by replacing the Fos/Jun interaction with the E. coli dihydrofolate reductase-trimethoprim interaction. I have demonstrated that this new split-Fluc based three-hybrid system can be used to profile kinase inhibitors in a variety of cell lines.

Vor dem Hintergrund der zunehmenden Prävalenz von Adipositas ist die Erforschung der zugrunde liegenden Pathomechanismen, unter anderem der Leptinresistenz, von großer Bedeutung. Der Schwerpunkt der vorliegenden Arbeit lag in der Etablierung und Validierung einer Methode zur Bestimmung der Bioaktivität von Leptin in Serum. Aufgrund des Vorhandenseins von Leptinbindungsproteinen erscheint nur der Teil des Serumleptins funktionell entscheiden, der tatsächlich den Leptinrezeptor erreicht und eine Bioantwort im Sinne einer Gewichtsreduktion auszulösen vermag. Der Bioassay basiert auf der Nutzung von Luciferase als Reportergen im HEK-293-Zellkulturmodell. Während der Validierung konnte gezeigt werden, dass der Test ein sensitives und spezifisches Verfahren zur Messung von Leptin im Serum darstellt. Problematisch blieb die Reproduzierbarkeit der Messergebnisse, so dass deren Interpretation im Vergleich mit Ergebnissen anderer Verfahren zum Nachweis von Leptin (Radioimmunoassay) sinnvoll erscheint. Der Luciferase-Bioassay wurde zur Bestimmung des Leptins im Serum adipöser Kinder eingesetzt, wobei eine statistisch signifikante Korrelation zwischen deren Bioaktivität und klinischen Markern der Adipositas gefunden wurde.

Plants have evolved a complex series of integrated defence mechanisms against pathogens. Following recognition of a pathogen avirulence (avr) gene product by the corresponding plant resistance (R) gene product, a complex signalling network is initiated. Local inducible defences are activated and a long-distance signal is released, leading to the establishment of systemic acquired resistance (SAR) to a wide range of pathogens. SAR is marked by the accumulation of pathogenesis-related (PR) proteins. Salicylic acid (SA) is a key signalling molecule in SAR, inducing PR gene expression both locally and systemically. In order to study further the molecular basis of SAR, we have developed a method of identifying novel SAR mutants by luciferase imaging. Transgenic Arabidopsis thaliana plants expressing a PR1a-luciferase reporter gene were generated and homozygous seed was chemically mutagenised. Mutants with perturbations in PR1 gene expression were identified and could be divided into various classes. A novel mutant expressing PR1 constitutively was selected for further study. cir1 (constitutively induced resistance 1) expressed both SA-dependent and SA-independent defence genes constitutively, accumulated SA to high levels and produced an increased amount of ethylene. In addition, cir1 exhibited resistance to the virulent bacterial pathogen Pseudomonas syringae pv tomato DC3000 and the virulent oomycete pathogen Peronospora parisitica Noco2. Genetic analyses indicated that cir1 is recessive and defines a mutation in a single gene, cir1 mapped to the lower arm of chromosome 4. Double mutants were produced between cirl and SA-, JA- and ethylene-insensitive mutants. Analysis of these plants showed that SA, JA and ethylene were required for constitutive defence gene expression and disease resistance in cir1. Thus, the results obtained indicate that CIR1 acts as a negative regulator in the disease resistance signal transduction network, most likely functioning upstream of the branchpoint between the SA-dependent and SA-independent pathways.

Eukaryotic protein kinases are one of the most important classes of human proteins, and a great deal of research has focused on the development of small molecule inhibitors as biological probes for the determination of their cellular function or as therapeutics for the treatment of disease, such as cancer. The need for new selective inhibitors and a better understanding of the selectivities of existing small molecules is readily apparent. Towards the goal of better understanding protein kinases and the molecules that inhibit them, I have developed a split-protein-based approach for the investigation of these kinase-small molecule interactions. Employing split-firefly luciferase as a reporter domain, we engineered a three-hybrid system capable of determining kinase inhibition through competitive interactions between an active site-directed ligand and a small molecule of interest. This method measures luciferase activity as a function of ligand binding, as opposed to the more traditional assays which quantify kinase activity directly, and alleviates the laborious process of protein purification. The model kinase PKA and the promiscuous ligand staurosporine were used in an initial test case to successfully validate the general design principles of our assay. The modular nature inherent to the assay's design enabled us to adapt it to roughly 300 additional protein kinases and two different ligands. We were able to establish a protocol for rapidly ascertaining the inhibition of a kinase by a library of 80 commercially available kinase inhibitors in a 96-well, high-throughput format. This protocol was then systematically applied to the AGC group of kinases to observe patterns of inhibition across similarly related kinases. We have further shown how these results might be correlated with the sequence identity between kinases to better anticipate inhibitor promiscuity. Finally, we were able to illustrate how a kinase-centric approach could be applied to correlate alterations to the kinase domain with changes in luminescence. This has use for the interrogation of different modes of inhibition as well as in identifying the specific determinants of inhibitor binding. In total, these efforts represent the optimization of a new, general platform for determining kinase inhibitor selectivity across the kinome, and it could potentially be applied universally to the interrogation of protein-ligand interactions.

Esta tese de doutorado descreve os estudos realizados para elucidar a biologia molecular da bioluminescência fúngica e sua relevância ecológica na natureza. A recente descoberta de que a luciferina fúngica é a 3-hidroxihispidina permitiu a caracterização do metabolismo secundário da fenilalanina nos genomas recém-sequenciados e transcriptomas de micélios das espécies luminescentes Panellus stipticus e Neonothopanus gardneri. Adicionalmente os genomas e transcriptomas de variedades não luminescente de P. stipticus e Lentinula edodes serviram como respectivos controles. Em geral, os genes envolvidos no metabolismo secundário da fenilalanina em amostras luminescentes tinham expressão igual ou superior àquela de espécies não luminescentes. Um agrupamento de genes relacionados com a biossíntese de fenilalanina foi encontrado em ambos os genomas luminescentes e não luminescentes de P. stipticus. A abundância de genes transcritos neste agrupamento foi semelhante para as espécies luminescentes e não luminescentes de P. stipticus, mas a policetídeo sintase tipo I em P. stipticus não luminescentes foi significativamente sub-regulada. Não foi encontrado agrupamento semelhante nos genomas de N. gardneri e L. edodes, sendo que os correspondentes homólogos estavam espalhados em diferentes loci. Extratos de fungos podem ser preparados in vitro, com a adição de 3-hidroxihispidina para produzir luz verde em abundância. A preparação de extratos proteicos de luciferase foi melhorada e a estrutura da luciferase, parcialmente purificada, foi investigada por espectrometria de massas. A presença de luciferase nos géis de purificação foi revelada usando-se luciferina e molécula similares à luciferina advindas de extratos de plantas. O nicho ecológico nas vizinhas de cogumelos bioluminescentes foi investigado de duas maneiras, armadilhas adesivas com cogumelos artificiais de acrílico, iluminados com luz LED verde e através da observação direta de cogumelos bioluminescentes com fotografia no infravermelho com lapso de tempo. Os estudos ecológicos foram conduzidos nos biomas da Mata Atlântica e da Mata dos Cocais, no Brasil. Baratas, aranhas, tesourinhas, grilo e vagalumes tec-tecs foram os animais mais comuns que interagiram com os cogumelos. Todos estes animais podem agir como dispersores de propágulos e, em alguns casos, como defensores dos cogumelos.
This PhD thesis describes the studies performed to elucidate the molecular biology of fungal bioluminescence and the ecological significance of the trait in the wild. The recent discovery that the fungal luciferin is 3-hydroxyhispidin has allowed for the characterization of phenylalanine secondary metabolism in the newly sequenced genomes and mycelium transcriptomes of luminescent Panellus stipticus and Neonothopanus gardneri, additionally the genomes and transcriptomes of a non-luminescent variety of P. stipticus and Lentinula edodes served as respective controls. In general the genes involved in phenylalanine secondary metabolism had greater or equal expression in luminescent samples than non luminescent. A cluster of genes related to the secondary metabolism of phenylalanine was found in both luminescent and non luminescent P. stipticus genomes. Transcript abundance of genes in this cluster was similar in both luminescent and non-luminescent Panellus stipticus, but the type I polyketide synthase in non luminescent Panellus stipticus was significantly down regulated. A similar gene cluster in the N. gardneri and L. edodes genomes was absent with corresponding homologues scattered at different genomic loci. Cell free fungal extracts can be combined in vitro with the addition of 3-hydroxyhispidin to produce abundant green light. Preparation of proteinaceous luciferase extracts was improved and partially purified luciferase samples were investigated by mass spectrometry. The presence of luciferase in the separation gel was also evidenced by using luciferin and luciferin-like molecules from plant extracts. The ecological niche surrounding bioluminescent mushrooms was investigated through two main means, glue traps with acrylic mushroom facsimiles that were internally illuminated with green LED lights and direct observation of bioluminescent mushrooms with infrared time lapse photography. Ecological studies were performed in the Atlantic rainforest (Mata Atlântica) and transitional Coconut Palm forest (Mata dos Cocais) biomes of Brazil. Cockroaches, spiders, earwigs, crickets, and luminescent click beetles were the most common animal interacting with mushrooms. All of these animals may be acting as fungal propagule dispersers and in some cases defense of the mushroom.

Die Induktion antioxidativer Enzyme gilt als eine Möglichkeit, die antioxidative Kapazität von Zellen zu steigern und dadurch mit oxidativem Stress assoziierten Erkrankungen (z. B. Herz-Kreislauf-Erkrankungen, Neurodegeneration, Atherosklerose) vorzubeugen. Ausgehend davon wurde in der vorliegenden Arbeit der Dual-Luciferase-Reportergen-(DLR)-Assay zum Nachweis der Induktion der antioxidativen Enzyme Katalase (CAT), zytosolische Glutathion-Peroxidase (GPX1) und Kupfer-Zink-Superoxid-Dismutase (SOD1) entwickelt. Im Zuge dessen wurden drei Säugetierzelllinien (CaCo2, IEC-18, V79) auf ihre Eignung zur Modellzelllinie untersucht. Aufgrund der Transfektionseffizienz wurde die Fibroblastenzelllinie V79 ausgewählt. Zur Gewährleistung eines hohen Substanzdurchsatzes des DLR-Assays wurden bei der Etablierung Parameter wie Kulturplattenformat, DNA-Menge, Luciferasen-Kinetik berücksichtigt. Nach erfolgreicher Etablierung des Versuchs im 96-Well-Format wurden L-Carnitin, Catechin, Epigallocatechingallat, Genistein, Wasserstoffperoxid (H2O2), Natrium-Ascorbat, Paraquat, Quercetin, 12-O-Tetradecanoylphorbol-13-Acetat (TPA) und Trolox in nicht-zytotoxischen Konzentrationen hinsichtlich der Aktivierung des Ratten-CAT-, des humanen GPX1- und des humanen SOD1-Promotors untersucht. Die Bestimmung der maximal tolerierbaren Behandlungskonzentration erfolgte im Vorfeld mittels Resazurintest. Von den zehn Verbindungen zeichneten sich drei Substanzen als potente Induktoren für die SOD1 und die GPX1 aus. Die 24-stündige Behandlung von mit Reportergenkonstrukten transient transfizierten V79-Zellen mit 100 µM Paraquat resultierte in einer Verdopplung der relativen SOD1-Promotor-Aktivität und einer Erhöhung der relativen GPX1-Promotor-Aktivität auf 1,6 bzw. 1,7. Die Stimulation mit 20 µM Genistein oder 10 µM Quercetin führte wiederum zu einer Verdopplung bis Verdreifachung der relativen SOD1- und GPX1-Promotor-Aktivität. Der Promotor der Rattenkatalase konnte demgegenüber nur durch 50 µM H2O2 aktiviert werden (1,5fach). Für diesen DLR-Assays bieten sich folglich Genistein, Quercetin wie auch H2O2 als Referenzsubstanzen an. Um aber eine qualitative Charakterisierung der einzelnen Verbindungen hinsichtlich ihres Induktionspotentials zu gewährleisten, sollten von allen getesteten Substanzen Dosis-Wirkungskurven aufgenommen werden. Zudem wird für den routinemäßigen Einsatz die Verwendung stabil transfizierter Zellen zur Vermeidung von mit der Transfektion verbundenen experimentellen Schwankungen empfohlen.
The induction of antioxidative enzymes might be an opportunity to elevate the cellular antioxidative capacity and, thus, to prevent oxidative stress associated diseases (e. g. cardio-vascular disease, neurodegenerative disease, atherosclerosis). Based on this idea the dual luciferase reporter gene (DLR) assay was developed to demonstrate the induction of three antioxidative enzymes: catalase (CAT), cytosolic glutathione peroxidase (GPX1), and copper-zinc superoxide dismutase (SOD1). In the course of the development three mammalian cell lines (CaCo2, IEC-18, V79) were tested for their ability to serve as a model cell line. The line V79 was chosen due to the transfection efficiency. To give consideration to a high-throughput several parameters were studied (e. g. format of the cultural plates, amount of DNA, kinetics of the luciferases) and the DLR assay was successfully established in 96 well plates. Subsequently, L-carnitine, catechin, epigallocatechin gallate, genistein, hydrogen peroxide (H2O2), sodium ascorbate, paraquat, quercetin, 12-O-tetradecanoylphorbol-13-acetate (TPA) and trolox were tested in non-cytotoxic concentrations for the activation of the rat CAT, human GPX1 and human SOD1 promoter. The maximally tolerable concentrations were determined by resazurin test in advance. Three out of these ten compounds were identified as potent inducers of GPX1 and SOD1. Stimulation of reporter gene construct transient transfected V79 cells for 24 hours with 100 µM paraquat caused a duplication of the relative GPX1 promoter activity and a 1.6-/1.7-fold increase of the relative SOD1 promoter activity. The incubation with 20 µM gen-istein or 10 µM quercetin resulted in duplication to triplication of both, the relative GPX1 and SOD1 promoter activity. In contrast, the rat CAT promoter was activated by 50 µM H2O2 (1.5-fold). Consequently, genistein, quercetin, and H2O2 are considered to be suitable reference substances for this DLR assay. To further characterize the inducing potential of the tested compounds all of them should be tested in different concentrations. Furthermore, for the routinely performed DLR assay it is recommended to use stably transfected cells to eliminate transfection caused variations.

Mycobacterium avium causes disseminated infection in humans with immunodeficiency, pulmonary infections in individuals with predisposing lung conditions (e.g., pneumoconiosis), and cervical lymphadenitis in children. Twenty-five to fifty percent of late stage AIDS patients are infected with M. avium. M. avium has been recovered from drinking water and strains from water share identical DNA fingerprints with isolates recovered from patients exposed to the water. Further, M. avium is resistant to chlorine, a disinfectant commonly used in municipal water supplies. Because of the slow growth of M. avium, measuring its susceptibility to disinfectants is laborious and reaction to a potential problem is delayed. Thus, there exists a need for a rapid test to measure the antimycobacterial disinfectant capability of chlorine containing water samples. The objective of this research was to develop a rapid and quantitative assay for the viability of mycobacteria using firefly luciferase as a reporter gene for disinfection survival studies. Derivatives of M. avium strains MD1 and A5, Mycobacterium smegmatis strain VT307 and Mycobacterium bovis BCG strain Pasteur carrying the firefly luciferase gene (pLUC10) were constructed. In pLUC10-carrying strains of M. avium strain A5 and M. smegmatis strain VT307, a direct correlation was shown between the quantity of light produced and the number of cells recovered as colony forming units. In disinfection studies of both pLUC10-carrying derivatives of M. avium strain A5 and M. smegmatis strain VT307, survival, as measured in colony forming units, correlated with survival in relative light units. Luciferase measurements appear to offer a method for rapid enumeration of mycobactericidal disinfection capacity of chlorinated water.
Master of Science

Despite the importance of molecular recognition in nearly all aspects of protein function, the determinants of specificity for enzyme-substrate and protein-protein interactions are poorly understood. The majority of these complexes involving bacterial luciferase from V. harveyi have yet to be fully characterized. Luciferase catalyzes the reaction of molecular oxygen, FMNH2 and a long-chain aliphatic aldehyde yielding FMN, the corresponding carboxylic acid and blue-green light. In vivo, luciferase is thought to obtain FMNH2 following transfer from a transiently associated oxidoreductase. To identify the oxidoreductase responsible for providing FMNH2 in E. coli, bioluminescence was compared using single gene deletion strains deficient in either a homolog to the endogenous V. harveyi oxidoreductase (Frp) or an oxidoreductase distantly related to luxG from V. fischeri (Fre). Fre is responsible for reducing flavin in vivo but does not physically interact with luciferase. The association between luciferase and the flavin product is also described. Luciferase was crystallized and subjected to soaking with high concentrations of FMN. A model was obtained for luciferase bound to FMN. Using molecular dynamics, models for the enzyme:aldehyde, enzyme:FMNH2, and luciferase bound to several reaction intermediates are presented. Finally, a conserved loop region adjacent to the active center was investigated for the ability to facilitate protein:protein interaction between luciferase and the endogenous Frp oxidoreductase. Following alanine mutagenesis of the charged residues throughout this loop, it appears that the residues targeted by this study are not components of a docking platform but facilitate a lid-gating mechanism of paramount importance for catalytic function.

O câncer compreende uma doença multifatorial responsável por altíssimos indíces de mortalidade globalmente. Embora atualmente tenhamos resultados positivos em relação ao tratamento do câncer principalmente voltados à imunoterapia, dados alarmantes ainda são encontrados. Assim, desenvolvemos linhagens tumorais geneticamente modificadas para expressarem ovalbumina (mOVA ou cOVA) e luciferase, a fim de estudarmos as interações do sistema imune com o tumor. Em nossos resultados, a presença da ovalbumina indicou: Alteração no perfil de crescimento tumoral em animais previamente imunizados com OVA e posteriormente desafiados com o tumor, ativação de células TCD8+ citóxicas anti-OVA além de demonstrar também a capacidade imunogênica das linhagens tumorais quando estas são administradas nos animais em estado necrótico. Ao todo, nosso modelo demonstrou que estratégias de vacinação anti-tumorais possuem a capacidade de ativação do sistema imune para na otimização do reconhecimento e resposta anti-tumoral.
Cancer is characterized as a multifactorial disease responsible for many deaths globally. Although nowadays we can find positive perspectives regarding cancers treatment, it is still very common to notice some alarming data. Therefore, our group developed some genetically modified tumoral lineages expressing ovalbumin (mOVA or cOVA) together with luciferase, in order to elucidate the relationship between tumor and the immune system. In our results, the presence of ovalbumin demonstrated: Changes in tumoral growth when animals were previously immunized with OVA and then challenged with our tumoral lineages; TCD8+ lymphocytes anti-OVA activation thus ovalbumin immunogenic potential when lineages were exposed to necrotic death followed by in vivo administration. In summary, our model showed that anti-tumoral vaccinations are indeed capable of promoting immune systems activation and consequently, improving the anti-tumor immunity.

Esta tese descreve estudos realizados na tentativa de purificar e caracterizar enzimas envolvidas na BL de fungos, além de trabalhos conduzidos a fim de investigar o mecanismo da bioluminescência de fungos. Inicialmente, tentou-se isolar as duas enzimas supostamente responsáveis pala reação bioluminescente em fungos. Parâmetros de atividade ótima (pH e temperatura) e comportamento cinético foram investigados. Todavia, com a descoberta de que a luciferina fúngica é o derivado hidroxilado da hispidina (3-hidróxihispidina), novas estratégias foram abordadas. Os esforços se concentraram na purificação da luciferase, visto que a hidroxilase não faz parte do sistema bioluminescente de fungos. Avaliação da interação da luciferase fúngica com a luciferina ou derivados dela sugeriram comportamento relativamente promíscuo da enzima. Os resultados indicaram que a reação luciferina-luciferase é favorecida em meio básico (pH ~8), a ~20 °C. Ensaios com 18O2 revelaram que a inserção de oxigênio na molécula de luciferina produz um intermediário cuja descarboxilação gera a oxiluciferina. Paralelamente, a síntese da hispidina in vitro a partir de ácido cafeico na presença de malonil-CoA e de extrato de micélios bioluminescentes resultou na emissão de luz, confirmando que a luciferina é reciclada no processo.
This work describes studies performed to purify and characterize enzymes responsible for the fungal bioluminescence. Also, it shows important data that contributes to understand the mechanism for bioluminescence reaction in fungi. First, we tried to isolate two enzymes suspected of being involved on fungal bioluminescence. Optimum activity parameters (pH and temperature) and kinetic behavior were investigated. However, the discovery that fungal luciferin is the hispidin derivative 3-hydroxyhispidin demanded adaptations in the project. First of all, concentrates efforts to luciferase purification was priority, since hydroxylase is not part of the bioluminescent system of fungi. Studies on the luciferase interaction with different substrates showed some promiscuity for the enzyme. The results indicated higher intensity of light from luciferin-luciferase reaction in alkaline solutions (pH ~ 8) at ~ 20 °C. The reaction in medium with 18O2 revealed that insertion of oxygen into the luciferin structure produces an intermediate whose decarboxylation generates oxyluciferin. In parallel, the in vitro synthesis of hispidin using caffeic acid and malonyl-CoA with the mycelium extract resulted in the emission of light, confirming that luciferin is recycled in the process.

La colonisation du tractus digestif (TD) par une bactérie requiert un ensemble de fonctions qu’il est important de connaître pour comprendre le fonctionnement de cet écosystème. Dans ce travail, nous avons étudié l’adaptation de Lactococcus lactis à l’environnement digestif. L. Lactis est un levain de l'industrie laitière et les développements récents ont démontré son potentiel comme vecteur pour délivrer des molécules à visée médicale. L’adaptation au TD a été étudiée sur des souris monoxéniques pour L lactis par une approche protéomique. L. Lactis s’établit à une population équivalente à celle de bactéries commensales. Le protéome montre la mise en place d’un catabolisme fermentaire original qui correspond à une adaptation à une carence en source carbonée. Nous avons montré qu’une protéine de fonction inconnue, YwcC, est essentielle au maintien de L. Lactis dans le TD et que cette protéine est une phosphogluconolactonase, impliquée dans la voie des pentoses phosphate. Nous avons aussi suivi l’adaptation de L. Lactis à l’ajout de lactose dans le régime des souris. Cet apport induit le catabolisme du lactose et réprime d’autres protéines, confirmant l’hypothèse de la carence carbonée comme étant un des paramètres-clé de l’environnement intestinal pour L. Lactis. La capacité à utiliser le lactose confère un avantage écologique important lorsque le sucre est présent dans l’alimentation. L’analyse protéomique comparative, la construction de mutants et l’utilisation d’animaux gnotobiotiques nous a permis d’appréhender la physiologie in vivo de Lactococcus lactis d’une manière globale et d’élargir nos connaissances sur des fonctions essentielles à la colonisation du TD
Knowledge of the bacterial functions required for colonization of the digestive tract (DT) is essential for the understanding of the properties of the microbiote. In this thesis, we studied the adaptation of Lactococcus lactis to the DT of monoassociated mice. L. Lactis is one the main starter used in the dairy industry and recent developments have demonstrated its potential as a living vehicle for the targeting of antigens or therapeutics. To identify the functions expressed by in the DT we used monoassociated animal model (mice) combined with a proteomic approach. L. Lactis is established at a population level equivalent to that of commensal bacteria. Proteome analysis indicated that the fermentation pathways activated in the DT by L. Lactis is reminiscent to that observed during carbon starvation. We identified YwcC, a protein of unknown function as essential for DT colonization. We showed that YwcC possesses a phosphogluconolactonase activity and is thus implicated in the pentose phosphate pathway. In a second study, we added lactose to the diet of L. Lactis monoassociated mice. The addition of the sugar induced the synthesis of lactose catabolic enzymes and repressed the synthesis of proteins involved in alternative catabolic pathways. This result confirmed the hypothesis of a carbon starvation physiological state for L. Lactis established in the DT. We observed that the presence of lactose provided a strong competitive advantage to strains able to catabolize it. By the combination of gnotobiotic animals, comparative proteome analysis and mutants construction, this thesis work provides novel informations on the in vivo physiology of Lactococcus lactis

Known luminous bacteria belong to the genera Vibrio, Photobacterium, Shewanella, and Photorhabdus. The enzyme luciferase catalyzing the luminous reaction is composed by the α and β polypeptides and subunit α is responsible for substrate binding and catalytic activities. Luciferases are classified into "slow " of Vibrio harveyi and "fast" of Photobacterium sps. on the basis of enzyme kinetics. Shewanella woodyi has "intermediate" kinetics. This research has tested the hypothesis of existence of three kinetic classes by sequencing luxA gene (coding for c subunit) of new strains and comparing these clusters to phenotypic analysis and sequencing of 16S rRNA. Phenotypic analysis has shown strains distinct from the known. LuxA amino acids and nucleotides and 16S rRNA sequences have shown 5 major lineages corresponding to known species. A dlade distinct from the known species was present. Geographic location and fish habitat didn't affect the distribution of strains.

[EN] Cells respond to environmental stimuli by fine tuned regulation of gene expression. In this thesis we investigate the dose dependent modulation of the genetic response upon nutrient and stress signals in yeast. A destabilized version of firefly luciferase was used in living yeast cells as a real-time reporter for gene expression. This highly sensitive and non-invasive system can be simultaneously used upon many different experimental conditions in small culture aliquots. This allows the dose-response behaviour of gene expression driven by any yeast promoter to be reported and can be used to quantify important parameters, such as the threshold, sensitivity, response time, maximal activity and synthesis rate for a given stimulus. We applied the luciferase assay to the nutrient-regulated GAL1 promoter and the stress-responsive GRE2 promoter. We find that luciferase expression driven by the GAL1 promoter responds dynamically to growing galactose concentrations, with increasing synthesis rates determined by the light increment in the initial linear phase of activation. The GAL1 gene is activated with continuously increasing synthesis rates in a well defined range of galactose concentrations, correlating with a dynamic increase of histone remodeling and subsequent association of the RNAPII complex. Dose dependent chromatin remodeling appears to be the basis for the dynamic GAL1 expression since mutants with impaired histone dynamics show severely truncated dose response profiles. In the case of the GRE2 promoter, we demonstrate that the very short-lived version of luciferase used here is an excellent tool to quantitatively describe transient transcriptional activation. The luciferase expression controlled by the GRE2 promoter responds dynamically to a gradual increase of osmotic or oxidative stress stimuli, which is mainly based on the progressive increase of the time the promoter remains active. In contrast, the GRE2 promoter operates like an off/on switch in response to increasing osmotic stress with almost constant synthesis rates and exclusively temporal regulation of histone remodeling and RNAPII occupancy. The Gal3 inducer and the Hog1 MAP kinase seem to determine the different dose response strategies at the two promoters. Our analysis reveals important differences in the way dynamic signals create dose sensitive gene expression outputs. Taken together, the luciferase assay described here is an attractive tool to rapidly and precisely determine and compare kinetic parameters of gene expression. Additionally, the function of the specific transcriptor factor Smp1 involved in the yeast osmostress response was investigated. Location analyses upon osmotic stress reveal that Smp1 associates preferentially with the whole transcribed regions (ORFs) upon stress as opposed to other transcriptional activators involved in the osmostress response. However, Smp1 seems to be important for stress-activated gene expression only in the presence of the natural induced gene and not of artificial promoter fusions. This highlights the possible role of Smp1 in regulating gene expression from ORF sequences rather than promoter regions.
[ES] Las células responden a los estímulos ambientales a través de una regulación precisa de la expresión génica. En este trabajo se investigó la modulación dosis dependiente de la expresión de genes activados en respuesta a estrés y por nutrientes. Se utilizó una versión desestabilizada de luciferasa de luciérnaga en células vivas de levadura como reportero para la detección de la expresión génica en tiempo real. Este sistema altamente sensible y no invasivo puede ser utilizado simultáneamente en diferentes condiciones experimentales a través de pequeñas alícuotas de cultivo. Esto permite la caracterización dosis-respuesta de la regulación de los promotores de levadura y puede ser utilizado para cuantificar parámetros importantes como el umbral, la sensibilidad, el tiempo de respuesta, la actividad máxima y el ratio de síntesis provocado por un determinado estímulo. Se aplicó el ensayo luciferasa al promotor GAL1 regulado por nutrientes y al promotor GRE2 activado en respuesta a estrés. Se observó que la expresión de la luciferasa activada por el promotor GAL1 responde de forma dinámica a las crecientes concentraciones de galactosa, con un incremento del ratio de síntesis determinado por el aumento de luz en la fase lineal inicial de la activación, en función de una gama de concentraciones de galactosa bien definidas. Este mecanismo de regulación depende de un aumento en la remodelación de las histonas y la consecuente asociación del complejo ARN pol II. La remodelación de la cromatina dosis dependiente parece ser la base de la expresión dinámica de GAL1, pues los mutantes relacionados con la dinámica de las histonas muestran perfiles dosis respuesta severamente afectados. En el caso del promotor GRE2, se demostró que una versión de una luciferasa desestabilizada es una herramienta excelente para describir de forma cuantitativa la activación transcipcional transitoria. La expresión de la luciferasa controlada por el promotor GRE2 responde de forma dinámica al aumento gradual de estímulo de estrés osmótico u oxidativo. La activación se observa principalmente en el incremento progresivo del tiempo en que el promotor permanece activo. Diferentemente de GAL1, el promotor GRE2 opera a través de un cambio apagado/encendido en respuesta a un aumento de estrés osmótico a través de ratios de síntesis prácticamente constantes y cuya regulación solamente depende de la remodelación de la cromatina y de la permanencia de la ARN pol II. Finalmente, se puede especular que el inductor Gal3 y la MAPK Hog1 son las moléculas determinantes para las diferentes estrategias de respuesta dinámica para los dos promotores. En este trabajo se identifican importantes diferencias en la señalización dinámica determinada por la dosis de estímulo en la expresión génica. En conjunto, el ensayo de luciferasa presentado en este trabajo puede ser una herramienta interesante para determinar y comparar de forma rápida y precisa los parámetros de la expresión génica. Adicionalmente se investigó la función del factor de transcripción Smp1 involucrado en la respuesta a osmoestrés en levadura. Un análisis de la asociación a la cromatina in vivo bajo estrés osmótico demostró que Smp1 se une preferentemente a regiones transcritas (ORFs) lo que refleja un comportamiento diferente comparando con otros activadores transcripcionales de la respuesta a estrés osmótico. Sin embargo, Smp1 parece ser importante para la expresión génica activada por estrés osmótico sólo en la presencia del gen natural inducido y no de fusiones artificiales del promotor. Esto evidencia el posible papel de Smp1 en la regulación de la expresión génica desde secuencias ORF y no en las regiones promotoras.
[CA] Les cèl·lules responen als estímuls ambientals a través d'una regulació precisa de l'expressió gènica. A aquest treball es va investigar la modulació dosi dependent de l'expressió de gens activats en resposta a estrès i per nutrients. Es va utilitzar una versió desestabilitzada de luciferasa de cuca de llum en cèl·lules vives de llevat com a reporter per a la detecció de l'expressió gènica a temps real. Aquest sistema altament sensible i no invasiu pot ser utilitzat simultàniament en diferents condicions experimentals a través de xicotetes alíquotes de cultiu. Això permet la caracterització dosi-resposta de la regulació dels promotors de llevat i pot ser utilitzat per a quantificar paràmetres importants com el llindar, la sensibilitat, el temps de resposta, l'activitat màxima i el rati de síntesi provocat per un determinat estímul. L'assaig luciferasa es va aplicar al promotor GAL1 regulat per nutrients i al promotor GRE2 activat en resposta a estrès. Es va observar que l'expressió de la luciferasa activada pel promotor GAL1 respon de forma dinàmica a les creixents concentracions de galactosa, amb un increment del rati de síntesi determinat per l'augment de llum en la fase lineal inicial de l'activació, en funció d'una gama de concentracions de galactosa ben definides. Aquest mecanisme de regulació depèn d'un augment en la remodelació de les histones i la conseqüent associació del complex ARN pol II. La remodelació de la cromatina dosi dependent sembla ser la base de l'expressió dinàmica de GAL1, ja què els mutants relacionats amb la dinàmica de les histones mostren perfils dosi-resposta severament afectats. En el cas del promotor GRE2, es va demostrar que una versió d'una luciferasa desestabilitzada és una eina excel·lent per a descriure de forma quantitativa l'activació transcripcional transitòria. L'expressió de la luciferasa controlada pel promotor GRE2 respon de forma dinàmica a l'augment gradual d'estímul d'estrès osmòtic o oxidatiu. L'activació s'observa principalment a l'increment progressiu del temps al qual el promotor roman actiu. De forma diferent de GAL1, el promotor GRE2 opera a través d'un canvi apagat/encès en resposta a un augment d'estrès osmòtic a través de ratis de síntesi pràcticament constants i als quals la seua regulació només depèn de la remodelació de la cromatina i de la permanència de l'ARN pol II. Finalment, es pot especular que l'inductor Gal3 i la MAPK Hog1 són les molècules determinants per a les diferents estratègies de resposta dinàmica per als dos promotors. A aquest treball s'identifiquen importants diferències a la senyalització dinàmica determinada per la dosi d'estímul a l'expressió gènica. En conjunt, l'assaig luciferasa presentat a aquest treball pot ser una eina interesant per a determinar i comparar de forma ràpida i precisa els paràmetres de l'expressió gènica. Addicionalment, es va investigar la funció del factor de transcripció Smp1 involucrat en la resposta a osmoestrès en llevat. Una anàlisi de l'associació a la cromatina in vivo sota l'estrès osmòtic va demostrar que Smp1 s'uneix preferentment a regions transcrites (ORFs), el qual reflecteix un comportament diferent comparant amb altres activadors transcripcionals de la resposta a estrès osmòtic. Tot i així, Smp1 sembla ser important per a l'expressió gènica activada per estrès osmòtic només en la presència del gen natural induït i no de fusions artificials del promotor. Això evidencia el possible paper de Smp1 en la regulació de l'expressió gènica des de seqüències ORF i no a les regions promotores.
Rienzo, A. (2016). Estudio de la regulación dinámica de la expresión génica en respuesta a estrés osmótico en levadura [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/62160
TESIS

Fireflies are beetles that generate yellow-green light when their luciferase enzyme activates and oxidizes its substrate, D-luciferin. This bioluminescent reaction is widely used as a sensitive reporter both in vitro and in vivo. However, the light-emitting chemistry is limited by the properties of the small molecule D-luciferin. Our lab has developed a panel of synthetic luciferin analogs that improve on the inherent characteristics of D-luciferin. My thesis work focuses on harnessing these novel substrates to further expand the utility and molecular understanding of firefly bioluminescence. The first part of my thesis focuses on using synthetic luciferins to improve bioluminescence imaging beyond what is possible with D-luciferin. Our substrates emit red-shifted light compared to D-luciferin, bringing the wavelength to a range that is more able to penetrate through tissue, but at a cost of lower signal intensity. I developed mutant luciferases that increase the maximal photon flux with the synthetic luciferins over what is achievable with the wild-type luciferase, and furthermore discriminate between substrates based on their chemical structures. Additionally, I have expanded the bioluminescence toolkit by harnessing the intrinsic properties of the luciferins to non-invasively and specifically assay the activity of a single enzyme (fatty acid amide hydrolase) in live mice. Therefore, my work presents an effective way to generally improve upon bioluminescent reporters, but also to measure the activity of a specific enzyme of interest in the context of a living organism. The second part of my thesis employs synthetic luciferins to more deeply probe the light-emitting chemistry of bioluminescence. Our synthetic substrates reveal latent luciferase activity from multiple luciferase homologs that are inactive with D-luciferin. These enzymes, the fatty acyl-CoA synthetases, are predicted to be luciferase’s evolutionary predecessors, but it was not clear how the light emitting chemistry originated. My work shows that the luciferase must activate the luciferin and provide oxygen access, but the light emitting chemistry is a fundamental property of that activated intermediate. In summary, the work described herein not only expands our understanding of firefly bioluminescence, but also broadens its practical applications to shine bioluminescent light on the dark corners of biology.

This dissertation examines the social history of Atlantic and American free and unfree labor by focusing on the production and consumption of the means of light from the colonial period to the end of the nineteenth century. Drawing from archives across the country, I reconstruct the ground-level experiences and struggles of the living (and dying) bringers of lights--those American lucifers--and the worlds they made in the process.
History

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Fundação Oswaldo Cruz. Centro de Pesquisas Aggeu Magalhães. Recife, PE, Brasil
O vírus da febre amarela (YFV, Yellow Fever Virus), um arbovírus da família Flaviridae,é o agente causador da febre amarela (FA), uma doença aguda, febril, não contagiosa, hemorrágica e potencialmente fatal. O YFV é endêmico em regiões tropicais da América do Sul e África. Apesar de sua significância como um problema de saúde pública, muitos mecanismos moleculares da biologia do YFV, como replicação do genoma e patogênese viral ainda não foram bem compreendidos. Avanços em genética reversa viral tem permitido a elucidação de mecanismos da biologia e comportamento viral, bem como a construção de vetores vacinais e desenvolvimento de drogas antivirais. No presente trabalho, descrevemos a construção e caracterização de um vírus recombinante de FA expressando o gene repórter da Gaussialuciferase (GLuc). Utilizando o sistema de recombinação homóloga em levedura, o gene repórter da Proteína Fluorescente Amarela (YFP, Yellow Fluorescent Protein) do vírus recombinante YFV-YFP-DENV1linker, previamente construído em nosso laboratório, foi substituído pelo gene repórter GLuc. A construção foi confirmada por PCR. Os RNAs virais genômicos foram sintetizados in vitro, e posteriormente transfectados em células BHK-21.As células transfectadas foram avaliadas por imunofluorescência indireta e mensuração do gene repórter GLuc. Dois clones foram recuperados e caracterizados em cultivo celular. Nós acreditamos que este vírus repórter deverá ser útilna triagem e desenvolvimento de drogas antivirais específicas, estudos de replicação virale competência vetorial, além da possível utilização como vetor viral vacinal