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.

A enzima peroxidase (HRP) , agindo como uma oxigenase em substratos apropriados, promove a formação de espécies excitadas no estado triplete. Estes produtos excitados podem ser formados em sistemas bioquímicos e promover processos fotoquímicos no escuro. Nesta linha de estudos, investigamos a oxidação aeróbica, na presença de HRP, de substratos contendo ligações de Schiff. Esses compostos são de grande importância biológica, uma vez que participam como intermediários em diversas reações enzimáticas (transaminação, biossíntese de aminoácidos, biossíntese de profirinas), nas ligações cruzadas do colágeno e da elastina, na hemoglobina AlC, na rodopsina e bacterionodopsina e como produtos finais da lipoperoxidação. A oxidação aeróbica de quatro iminas alifáticas (BPA, i-BMA, sec-BMA e BVA) , catalisada por HRP, é quimiluminescente. A natureza triplete da espécie excitada foi sugerida pelo espectro de quimiluminescência, por meio de estudos de transferência de energia para DBAS e clorofila e pela supressão de quimiluminescência por oxigênio, sorbato, indol e p-benzoquinona. Com base nos altos valores de kETo encontrados, concluimos que a transferência de energia esteja ocorrendo provavelmente por um mecanismo a longa distância. A análise dos produtos da reação e os dados cinéticos indicam que a reação ocorre provavelmente segundo a via: (VER Esquema no arquivo) Ressalta-se que esta reação constitui um modelo mais adequado para sistemas bioluminescentes do que o proposto por McCapra e Burford (1976) com bases de Schiff aromáticas em t-butóxido e DMSO. Os sistemas aqui descritos também apresentam a possibilidade de desenvolvimento de método analítico, empregando quimiluminescência, para detectar formação de bases de Schiff em sistemas biológicos. Os estudos com base de Schiff alifáticos (sistemas modelo) foram estendidos para adutos contendo ligação de Schiff entre glicolaldeído e aminoácidos (Lys, Arg, His e Phe) ou proteínas (lisozima, BSA e protaminas). Todos os sistemas estudados são quimiluminescentes na presença de HRP; a fluorescência característica da formação do aduto decai concomitantemente com a emissão de luz; e, durante a reação, a peroxidase encontra-se principalmente na forma de composto II. Observa-se também transferência de energia dos sistemas glicolaldeído-lisozima/HRP/O2 e glicolaldeídoprotamina/HRP/O2 para clorofila. Estes estudos têm grande interesse do ponto de vista da toxicidade associada à ingestão de álcool etílico, a qual, segundo trabalhos recentes da literatura, é atribuída à formação de bases de Schiff entre proteínas de membranas e acetaldeído. E notável também, a formação de iminas durante o processo de lipoperoxidação e a quimiluminescência que o acompanha. Nossos dados levantam a possibilidade de que a toxicologia do álcool pode envolver espécies eletronicamente excitadas formadas na oxidação aeróbica dos adutos aldeído-proteínas. / Horseradish peroxidase (HRP) , acting as an oxigenase on various substracts, promotes the generation of electronically excited triplet species. These species can also be formed in many biochemical reactions and drive photochemical processes in the absence of light. Enlighted by this hypothesis (photobiochemistry in the dark) we have investigated the HRP-catalyzed aerobic oxidation of substrates containing Schiff linkage. These compounds are of utmost biological importance as they participate as intermediates of several enzymatic reactions (transamination, amino-acid biosyntheses, porphyrin biosyntheses, a.s.o.), in the crosslinking of collagen and elastin, in hemoglobin AlC, in rhodopsin and bacteriorhodopsin, and as final products from lipid peroxidation. The aerobic oxidation of four aliphatic imines (BPA, i-BMA, sec-BMA and BVA) , catalyzed by HRP, has been shown to be chemiluminescent. The triplet nature of the products is suggested by the chemiluminescence spectrum, by energy transfer studies to DBAS and chlorophyll a and by quenching of the chemiluminescence by molecular oxygen, sorbate ion, indol and p-benzoquinone. Based on the high values of ETTo found in these experiments we have concluded that the energy transfer process occur through a long range mechanism. Analyses of the products found in the spent reaction mixtures and the kinetic data indicate that the reaction follows the route: (SEE scheme file) We stress that this reaction constitutes a more realistic model for bioluminescent systems than that reported by McCapra and Burford (1976), which uses aromatic Schiff bases in DMSO/t-butoxi. In addition, the reaction described here point out for the possible development of chemiluminescent analytical procedure to detect the formation of Schiff bases in biological systems. Our studies on aliphatic Schiff bases were extended to adducts of both amino acids (Lys, Arg, His and Phe) and proteins (lyzozyme, bovine serum albumin and fish protamins) with glycolaldehyde. All these adducts are chemiluminescent when exposed to HRP in an aerated buffered solution. The fluorescence typical of the adducts decays concomitantly with light emission and, during the reaction, the enzyme remains predominantely in the form of HRP Compound II. Energy transfer to chlorophyll a from the systems glycolaldenyde-lyzozyme/O2/HRP and glycolaldehyde-protamine/ 02/HRP occur. These studies are relevant with respect to the toxicity associated to ethyl alcohol intake which, according to recent reports, is attributed to the formation of Schiff linkages between membrane proteins and acetaldehyde. Also noteworthy is the formation of imines along lipid peroxidation and the accompanying chemiluminescence. Our data raise the hypothesis that the alcohol toxicology may involve generated electronically excited species formed during aerobic oxidation of protein-aldehyde adducts.

A bioluminescência desperta o interesse humano há muitos séculos. Presente em quatro dos sete reinos taxonômicos, Monera, Chromista, Animalia e Fungi, cada um com mecanismos muito diferentes. Pode-se dizer que o estudo químico da bioluminescência começou com os experimentos de Dubois no século XIX, que cunhou os termos luciferina e luciferase, termos genéricos para o substrato e enzima envolvidos na reação, respectivamente. No caso específico de fungos, o envolvimento de enzimas foi debatido por quase cinco décadas, após a proposta enzimática de Airth e Foerster na década de 1960 e a não enzimática por Shimomura, em 1989. Somente em 2009 a hipótese de Airth e Foerster foi confirmada pelo nosso grupo, seguido da identificação da luciferina fúngica e o envolvimento de hispidina, como molécula precursora, em 2015 pelo grupo de Yampolsky. Para conseguir elucidar mecanismos químicos, a técnica de espectrometria de massas pode ser empregada para a identificação estrutural de reagentes e intermediários destas e de outras reações orgânicas. Após a confirmação do envolvimento de hispidina na bioluminescência de fungos, utilizou-se a técnica de cromatografia líquida acoplada a espectrometria de massas para identificar a presença de hispidina, seus derivados e intermediários precursores, em fungos e em algumas plantas. / Bioluminescence arises human interest for centuries. Occurring in four of seven taxonomical Kingdoms, Monera, Chromista, Animalia and Fungi, each of them with completely different mechanisms. The chemical study of bioluminescence starts in XIX century with Dubois, who coined the terms luciferin and luciferase, generic terms for substrate and enzyme involved in the bioluminescent reaction, respectively. In the specific case of fungi, enzyme involvement has been debated for almost five decades, after the enzymatic proposal by Airth and Foerster, during the 1960 decade, and the non-enzymatic proposal by Shimomura in 1989. It was only in 2009 when the proposal by Airth and Foerster was confirmed by our group, followed by the identification of the fungal luciferin and the involvement of hispidin, as the precursor molecule, in 2015 by Yampolskys group. To elucidate the chemical mechanisms, mass spectrometry can be employed to structural identification of reagents and intermediates on these and other organic reactions. After the confirmation of hispidin involvement in fungi bioluminescence, liquid chromatography coupled with mass spectrometry was uses do identify the presence of hispidin, its derivatives and precursor intermediates in fungi and selected plants.

Non-viral gene delivery is plagued by low transfection levels compared to viral delivery. The nuclear envelope presents a significant obstacle for non-viral vectors. A peptide-based nuclear localizing sequence has been incorporated into non-viral vectors to traverse the nuclear envelope. Here, we selected a photo-chemical method for covalently labeling the peptide onto plasmid DNA. The hypothesis of this work was to incorporate a nuclear localizing sequence into a non-viral delivery vector, demonstrate increased nuclear uptake and show a subsequent increase in transgene expression both in vitro and in vivo. We focused on pursuing in vitro and in vivo methods by which to test non-viral vectors for increases in gene expression based on the nuclear localizing sequence. Hydrodynamic dosing and intramuscular dosing (followed by electroporation) are two efficient delivery routes for dosing DNA in vivo. Through preliminary experiments, we became confident that whole animal bioluminescent imaging was a reliable and quantitative method by which to detect luciferase expression by either delivery route. Moving forward, both hydrodynamic and intramuscular dosing would be used to test formulations for nuclear localizing ability in vivo. Nuclear localizing peptides containing a photo-activatable functionality were synthesized and characterized. We quantitatively explored the photo-labeling capabilities on plasmid DNA via a radioactive peptide. In vitro, tissue culture-based experiments were carried out to show increased nuclear uptake by confocal microscopy as well as increased transgene expression. Throughout the literature, achieving an increase in expression by incorporating a nuclear localizing sequence into a non-viral vector has been elusive. The complexity of achieving this goal is increased when considering an in vivo system for improving gene transfer efficiency. Several strategies have been explored to demonstrate an increase in reporter gene expression from this type of non-viral vector, and the methods developed herein can be applied to other nuclear localizing vectors.

Since its characterization, the definitive shortcoming of the bacterial luciferase (lux) bioluminescent reporter system has been its inability to express at a functional level in the eukaryotic cellular background. While recent developments have allowed for lux function in the lower eukaryote Saccharomyces cerevisiae, they have not provided for autonomous function in higher eukaryotes capable of serving as human biomedical proxies. Here it is reported for the first time that, through a process of poly-bicistronic expression of human codon-optimized lux genes, it is possible to autonomously produce a bioluminescent signal directly from mammalian cells. The low background of the bioluminescent signal, along with its characteristic lack of substrate amendment required for bioluminescent production, makes a mammalian-based lux reporter system ideal for real-time monitoring of cell culture or murine model systems. The delectability of a lux-based system provides for a functionally equivalent process to monitoring firefly luciferase-expressing cells under cell culture or subcutaneous imaging conditions without the well-documented uncertainties stemming from additional substrate introduction. However, the relatively blue-shifted emission wavelength of the lux reporter system, along with its low quantum yield, has been shown to reduce its effectiveness for use during deep tissue imaging of animal subjects. Despite these disadvantages, it has been demonstrated that a human cell line expressing the human codon-optimized lux genes can function as a biosensor for determination of human bioavailability of toxic compounds and that, by regulating the production of the luxC and luxE genes, the lux system can be employed as the first mammalian, real-time, fully autonomous bioreporter. These cell lines provide unique and efficient models for the detection and monitoring of human-relevant compounds of interest. The limiting reagent for bioluminescent production in the mammalian cellular background has been determined to be the cytosolic availability of the FMNH2 co-substrate and, in light of this evidence, directions for future optimization have been characterized and evaluated in respect to their ability to increase bioluminescent yield under these conditions.

Chez la majorité des organismes vivants, depuis certaines bactéries, jusqu'aux mammifères, de nombreux processus biologiques sont rythmés, comme par exemple l'alternance veille sommeil, ou encore le cycle d'hibernation. Le contrôle de ces rythmes biologiques est effectué grâce à des horloges biologiques, qui sont dans certains cas des oscillateurs auto-entretenus programmés génétiquement. Les oscillateurs biologiques dont la période est de proche de 24h sont dits circadiens. La cyanobactérie Synechoccocus elongatus sp. PCC7942 est l'un des plus simples organismes possédant cette horloge circadienne. Malgré la division cellulaire (jusqu'à 3 divisions par cycle), l'oscillation circadienne persiste au sein d'une population. De plus, des mesures faites sur cellules individuelles montrent la persistance de l'oscillateur circadien avec un temps de corrélation de plusieurs mois. Nous nous intéressons ainsi à l'origine de la robustesse de l'oscillateur : comment conserver une oscillation aussi stable malgré les sources de bruits (fluctuations internes et environnementales) auxquelles est soumis le réseau génétique ? Une telle stabilité pourrait être intrinsèque à l'oscillateur, ou alors être due à une interaction entre oscillateurs. Notre étude s'intéresse à la 2ème hypothèse ; ainsi, nous avons cherché l'existence potentielle d'un couplage entre 2 populations d'oscillateurs circadiens. Pour accéder à l'oscillation de l'horloge circadienne, nous avons utilisé une souche sauvage dans laquelle a été introduit le gène rapporteur de la luciférase. La souche mutée ainsi obtenue va présenter une luminescence oscillante, traduisant l'expression de la luciférase sous le contrôle du promoteur correspondant. Les souches mutées et non mutées ont une activité génétique régulée par l'horloge circadienne. L'interaction est étudiée en réalisant des mélanges de deux populations -souche mutante +souche sauvage- de cyanobactéries ayant initialement des phases d'oscillation différentes, avec un rapport de concentration 1:20. L'évolution temporelle de la bioluminescence donne ainsi accès à l'oscillation circadienne de la population minoritaire. Les éventuels effets du couplage sont analysés en étudiant la phase d'oscillation de la population minoritaire. Notre étude a montré qu'après une quarantaine de jours d'interaction potentielle, la phase d'oscillation des minoritaires n'est pas affectée de façon significative par la présence d'oscillateurs ayant une autre phase initiale. Au vu de ces résultats, nous avons établi un modèle théorique de couplage afin de majorer l'éventuel couplage entre oscillateurs cyanobactériens. Notre modèle est principalement décrit par le modèle de Kuramoto, qui donne une description pertinente de nombreux oscillateurs biologiques. Des simulations numériques ont montré qu'il était possible de faire des hypothèses simplificatrices concernant la forme des distributions de phase au sein de chacune des populations en interaction. Ainsi, nous avons établi une relation simple entre le changement de phase, et la constante de couplage. En tenant compte des incertitudes sur la phase calculée expérimentalement, nous estimons une borne supérieure de la constante de couplage entre oscillateurs cyanobactériens.

Thèse de doctorat : Sciences de la vie et de la santé : Tours : 2008. / Titre provenant de l'écran-titre.

Reproduction de : Thèse de doctorat : Biochimie-biophysique cellulaire : Toulouse 3 : 2007. / Titre provenant de l'écran-titre. Bibliogr. p. 161-174.

Thesis (Ph. D.)--University of Hawaii at Manoa, 2003. / Includes bibliographical references (leaves 162-167). Also available via World Wide Web.

Biosensors are extremely powerful analytical tools instrumental for detection and quantification of bio-molecules such as DNA, peptides and even metabolites. The recent decade has seen a surge in biosensing applications ranging from molecular diagnostics, environmental monitoring, basic life science research, forensics and biothreat monitoring. The existing biosensor systems of today, however, have several limitations. They are expensive, bulky in size, power hungry, hard to use and with access limited to core facilities. Among other disadvantages, these impediments discourage the availability of point-of-care testing and low cost in-vitro diagnostics (IVD) in locations such as developing and third world countries. The main bottleneck in the development of low-cost and compact biosensors is the effective and efficient integration of several complex components present inside a typical biosensor. These components are the sample preparation, biomolecular recognition, signal transduction and data analysis. With vii the recent advancements in very large scale integration (VLSI) and fabrication technologies, it is now possible to integrate several of these biosensing components into a small form factor. This thesis proposes leveraging the utilization of VLSI technology to develop a low-cost, miniature, portable, fast analysis, high throughput and low power consumption biosensor solution. Apart from the miniaturization bene- fits, employing VLSI technology facilitates low-cost, high yield and low process variation. We present complementary metal-oxide semiconductor (CMOS) integrated microsystem solutions for fluorescence, bioluminescence and electrochemical biosensing. Simulation models are provided for the microsystems and the specifications for the constituent components derived. A common problem in the transducer development of biosensors that we specifically focus on, is the presence of a large non-informative signal called the background signal. This background signal can be several orders of magnitudes higher than the signal of interest and it reduces the overall sensitivity of the biosensor. Existing transducer solutions rely on very high dynamic range, expensive and power hungry solutions to solve the problem of high background signal. To address the problem of overwhelming background signal, this thesis proposes an active background subtraction architecture merged with a Σ∆ modulator. The robust, versatile architecture can be conveniently employed for optical and electrochemical sensing. The proposed architecture attenuates the background signal very early in the signal chain, achieving high dyviii namic range while significantly relaxing the performance requirements of the subsequent circuit blocks in terms of power dissipation, area and bandwidth requirements. To validate the proposed solution, two CMOS IC prototypes were developed for optical and electrochemical sensing respectively. A 12 × 12 array of Σ∆ photodetector with in-pixel background subtraction was developed in 0.18µm standard CMOS technology. The pixel performance has been validated with over 140dB dynamic range and the ability of subtract the background subtraction current validated from 10nA to 10fA. Real time pyrosequencing experiment has also been performed utilizing the photodetector array. A 12 × 12 array of Σ∆ electrochemical sensor with in-pixel background subtraction was developed in 0.18µm standard CMOS technology. Capacitive charge redistribution circuit architecture for bipolar current measurements was employed. The circuit performance was validated over the wide input current range of 100nA to 1pA. / text