Dans la recherche de thérapie antitumorale de plus en plus innovante, nous avons évalué un traitement local basé sur l'utilisation de plasma froid. Le plasma froid (dans ce cas, <40°C) est un gaz ionisé par un apport d'énergie. Il contient des charges (électrons, ions), des radicaux libres et des molécules excitées. Il peut être généré à l'extrémité de cathéter permettant un traitement locorégional comme le traitement de dysplasie ou encore de tumeurs non résécables. Une activité antitumorale importante du plasma a été mise en évidence in vitro sur diverses lignées tumorales (colorectale, pulmonaire, pancréatique et cérébrale). Par ailleurs les cellules tumorales sont plus sensibles au plasma que les cellules normales. Les ROS générés sont à l'origine des principaux mécanismes d'action du plasma. Ils induisent de nombreux dommages à l'ADN, suivi d'un arrêt du cycle cellulaire conduisant à l'apoptose des cellules. Les études de tolérance ont mis en évidence l'innocuité de faibles doses de plasma sur le tissu traité permettant de définir les doses de plasmas utilisable dans le cadre de traitements antitumoraux. En utilisant des tumeurs xénogreffées en sous cutané et l'imagerie de bioluminescence, une activité antitumorale du plasma froid a été mise en évidence pour la première fois in vivo avec une augmentation de la survie des souris traitées d'environ 60%. Le traitement induit un arrêt de la prolifération tumoral avec une induction d'apoptose dans l'ensemble de la tumeur sans augmenter la surface de nécrose. L'effet antitumoral a également été démontré en utilisant le plasma gun sur un modèle de tumeurs colique et pancréatique en situation orthotopique chez la souris avec une augmentation de la survie (115%) accompagné d'une diminution de la métastasie. Ces résultats obtenues dans une démarche de recherche translationnelle montrent l'intérêt potentiel du plasma comme nouvel agent antitumoral.

Cette thèse est consacrée au traitement d'images de bioluminescence chez le petit animal. Ce type d'imagerie, bien qu'utilisé en routine pour la recherche en cancérologie par exemple, présente néanmoins des problèmes liés aux phénomènes de diffusion et d'absorption par les tissus internes à l'animal. Il s'ajoute à cela le bruit du système d'acquisition ainsi que le bruit lié aux rayonnements cosmiques. Ceci influe sur la qualité des images acquises et rend leur exploitation délicate. Le but de cette thèse est de compenser ces effets perturbateurs. Les travaux menés ont abouti à la proposition d'un modèle de formation des images de bioluminescence ainsi qu'à une chaîne de traitement adaptée composée d'une étape de filtrage suivie d'une étape de déconvolution. Après étude de la nature des différents bruits liés à l'acquisition, nous avons mis au point un nouveau filtre médian pour la suppression du bruit impulsionnel aléatoire présent sur les images acquises ; ce filtre représente le premier bloc de la chaîne proposée. Pour l'étape de déconvolution, nous avons mené une étude comparative de différents algorithmes de déconvolution. Cela a conduit à choisir un algorithme de déconvolution aveugle initialisé avec la réponse impulsionnelle estimée du système d'acquisition. Nous avons validé notre approche globale en comparant les résultats à la réalité terrain. Au travers de différents essais cliniques, nous avons montré que le traitement que nous proposons permet une amélioration significative de la mesure des sources bioluminescentes et une meilleure distinction de sources très proches, ce qui représente un apport non négligeable pour les utilisateurs d'images de bioluminescence.

Current filter validation methods require 48 hours culture for results to become available, which creates time delays within the manufacturing process and quality control back-logs The thesis compares alternative methods for the production of filter challenge test data Within 24 hours to the desired test sensitivity, using bioluminescent and fluorescent genetically engineered strains of the test organism Brevundzmonas dzmznuta The recombinant strains were produced using a Tn5 transposon system, using a filter mating method. The genes cloned into the bacterial chromosome were the biolummescence lux_ genes, taken from the marme bacteria, Photorhabdus lummescens or Vzbno harveyz, and the gene encoding green fluorescent protein taken from the marine jelly fish Aequoria victoria The cloned strains were found to show no difference to the w1ld type strain With respect to their surface hydrophobicity, according to a bacterial adherence to hydrocarbons assay, and surface charge, according to an electro-static interaction chromatography method. Furthermore, the cell size according to Transmission Electron Microscopy was not significantly different to the wild type strain, which had cell dimensions of 1 05 x 0 52 Jlm The retention of cells by 0 45 mtcron rated filters, was shown to be not significantly different to the wild type All strains were retained by 0 2 Jlm filters These data confirmed that the cloned strains were suitable for challenge testing Four methods were used to detect microcolonies of the recombinant strains on filters. The advantage of the microcolony detection system was that it showed that the cells detected downstream of the filter were viable and culturable. The best detection method was with an epifluorescent microscope and the fluorescent strain after 24 hours, for which the sensitivity was 98.1 %. Two CCD camera systems were used to detect the bioluminescent strains on filters. The sensitivity of these systems were 80.1% and 83 9%, for the Nucleovision and Nightowl CCD camera systems, respectively, after 24 hours In addition, the Bwprobe photomultipherbased system was shown to achieve the detection sensitivity of one microcolony after 24 hours. Also, steps were made to study transcription Initiation signals for gene expression in fluorescent recombinant Brevundzmonas dzmmuta. Various putative promoter sequences were Identified in one fluorescent strain, using a DNA sequencmg method. These sequences showed homology to previously identified E colr and Brevundzmonas promoter sequences. Finally, an attempt was made to produce recombinant fluorescent and bioluminescent Acholeplasma lazdlawu, however this was unsuccessful and further work will be required to achieve this objective.

Thesis (M.S. in Biomedical Engineering)--Vanderbilt University, May 2005. / Title from title screen. Includes bibliographical references.

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.

Este estudo foi realizado com o objetivo de desenvolvimento e validação do método microbiológico rápido empregando a técnica de bioluminescência de Adenosina Trifosfato (ATP) como método alternativo para o teste de esterilidade. O ATP reage com o sistema enzimático luciferina/luciferase e gera um fóton de luz em presença de íons magnésio, reação que pode ser utilizada na detecção de microrganismos. A luz gerada na reação é medida por um dispositivo chamado luminômetro, que traduz o sinal em unidades relativas de luz (URL), metodologia altamente sensível que pode ser utilizada na análise de produtos estéreis com o objetivo de diminuição no tempo do ensaio. Enquanto a turbidez do meio de cultura só pode ser visualizada quando o contaminante chega à concentração de 106 UFC/ml, a tecnologia de bioluminescência de ATP pode detectar amostras com concentração em torno de 104 UFC. Foram empregados na validação dados obtidos a partir do método tradicional de esterilidade (técnica de filtração) realizado paralelamente ao método alternativo. As soluções parenterais utilizadas nos ensaios foram: solução fisiológica 0,9%; solução de dextrose 5%; ringer lactato; e solução de metronidazol 0,5%. As soluções-teste foram inoculadas intencionalmente com suspensões microbianas preparadas através da diluição de Bioballs&#174, com concentrações de 10 UFC/100 ml, 2 UFC/100 ml e 0,4 UFC/100 ml. Após a realização do teste convencional, as membranas resultantes do ensaio foram incubadas nos meios de cultura caldo caseína de soja e tioglicolato. Alíquotas destes meios foram retiradas após 96 horas de incubação para análise pelo método alternativo. Os seguintes microrganismos foram selecionados para o estudo: Staphylococcus aureus, Bacilus subtilis, Pseudomonas aeruginosa, Candida albicans, Clostridium sporogenes, Aspergillus brasiliensis, Kocuria rosea e Micrococcus luteus. A análise dos resultados obtidos mostrou que o método alternativo é capaz de detectar os microrganismos testados. Quanto à sensibilidade, o método alternativo apresentou vantagem na concentração 2 UFC/100 ml, e equivalência nas outras concentrações. A não interferência dos diferentes produtos e meios nos resultados encontrados permite vislumbrar evidência de robustez do método. Adicionalmente, em relação ao tempo de resposta, o método alternativo demonstrou ser equivalente ao convencional (p-valor=0,43). / This study is being conducted with a goal of validating and developing the fast microbiological method of ATP bioluminescence, as an alternative method to the sterility test. The ATP reacts with the enzymatic system luciferin-luciferase and produces light in the presence of magnesium ions, this reaction can be used for microorganism\'s detection. The light generated in this reaction can be measured by a device called luminometer that translates the signal in relative light units (RLU). This methodology has high sensibility and it can be used in the sterile products analysis with the objective of reducing the time of the sample. While the turbidity of the culture medium it can be visualized just when the sample reaches a concentration of 106 UCF per mL, the bioluminescence assay can detect samples at concentrations around 104 UCF per mL. The both methods, conventional (filtration technique) and alternative were done in parallel and the result data were used in the validation study. It was used in the assay the next parenteral solutions: physiological solution 0,9%, metronidazole solution 0,5%, dextrose solution 5% and Ringer lactate. The test solutions were inoculated with the microorganism suspensions, prepared by the dilution of the Bioballs® with result concentrations of 10 CFU/100 mL, 2 FU/100 mL and 0,4 CFU/ mL. After the conventional test was performed, the result membranes were incubated in thioglicollate and soybean casein broth. After 96 hours of incubation, aliquots from the broth were taken to perform the analysis by the alternative method. The following microorganisms were selected to perform the validation study: Staphylococcus aureus, Bacilus subtilis, Pseudomonas aeruginosa, Clostridium sporogenes, Candida albicans, Aspergillus brasiliensis, Kocuria rosea and Micrococcus luteus. The analysis of results shows that the alternative method can detect the test microorganisms. Regarding of the sensibility, the alternative method shows advantage in the 2 CFU/mL inoculum concentration and equivalence in the other two concentrations. The method shows evidence of robustness because the results were not affected by the products or culture media used in the assay. Additionally concerning the detection time, the alternative method was established equivalent to the conventional method (p-value=0,43).

Vibrio harveyi is a bioluminescent bacterium and the organism in which quorum sensing was discovered. It was recently found that a class of molecules, cyclic dipeptides, may be a new kind of quorum sensing signal that may affect other species in the genus. The purpose of this study was to determine if V. harveyi produced one of these molecules, cyclo(Phe-Pro) or cFP, and the effects it has on bioluminescence, growth and gene expression. Electrospray Mass Spectrometry was used to detect cFP, and it was found. While growth and gene expression were not significantly affected by cFP, bioluminescence was slightly induced at low concentrations. It appears that V. harveyi does not produce cFP and it does not significantly affect the luminescence quorum sensing controlled genes, and is most likely not a true signal, in V. harveyi.

Photocatalysis is a robust synthetic tool capable of breaking and assembling chemical bonds using single electron chemistry. This is achieved through the catalytic conversion of light energy to chemical energy in situ, such that the photons being delivered are themselves reagents. Herein, an inexpensive and environmentally-benign platform for scaling up photocatalytic reaction is disclosed, harnessing blue light naturally emitted by deep-sea bioluminescent bacteria. Photobacterium angustum GB-1 was demonstrated to photoexcite both polypyridyl organometallic chromophores and organic dyes at short molecular distances, enabling photocatalysis without any external energy-consuming lamps.While improving the eco-friendliness of photocatalysis itself, we also present a method to use photocatalysis for environmental remediation. Using visible light, a nontoxic organic photosensitizer, and oxygen, we demonstrate the controlled oxidative depolymerization of polystyrene—including polystyrene retrieved from waste receptacles in Havemeyer—to acetophenone. This method is based on results obtained in the controlled aerobic deannulation of cycloalkanes, which is also discussed herein. Lastly, a means by which catalysis itself can be made more cost, resource, and time effective is presented. An innovative computational platform in development predicts new catalysts for reactions currently energetically inaccessible. In collaboration with the developers, we present experimental validation of their theoretical predictions, as well as perform the synthesis of a de novo fluorinated thiazolium precatalyst calculated to significantly lower the energetic barrier of an otherwise energetically prohibitive Stetter reaction.

No description available.