Thesis (Ph. D. in Cell and Developmental Biology)--Vanderbilt University, Dec. 2004. / Title from title screen. Includes bibliographical references.

Orientador: Clarice Queico Fujimura Leite / Banca: Lucilaine Ferrazoli / Banca: Carmo Elias Andrade Melles / Banca: Wagner Vilegas / Banca: Antonio Carlos Massabini / Resumo: O aumento de cepas de Mycobacterium tuberculosis resistentes às drogas utilizadas no esquema de tratamento convencional, principalmente por falha terapêutica, têm levado os pesquisadores à busca de novas drogas. Entretanto, faz-se necessário desenvolver novas metodologias para a determinação da atividade bactericida destes compostos. Este estudo descreve a padronização de uma metodologia rápida para triagem de atividade intra e extracelular de novos compostos, empregando Mycobacterium tuberculosis H37Rv ATCC 27294 e Mycobacterium tuberculosis Erdman ATCC 35801, ambas contendo o plasmídio pSMT1 com o gene luxA e luxB proveniente do Vibrio harveyi. A rifampicina e a isoniazida foram empregadas como droga de referência na padronização da técnica da luciferase extracelular, obtendo-se resultados de Concentração Inibitória Mínima de 0,03 μg/mL, para ambas as drogas, valores estes compatíveis com os da técnica de Alamar Blue. Para a padronização da técnica da luciferase intracelular foi utilizado o M. tuberculosis Erdman ATCC 35801 contendo o plasmídio pSMT internalizado em células de macrófagos J774. A droga de referência empregada foi a rifampicina obtendo-se um...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: There has been an increase of Mycobacterium tuberculosis strains that are resistant to the current anti-TB agents, mainly through acquired resistance by therapeutic failure. This fact has underscored the need of a quick development of antimycobacterial drugs that are more effective than those currently in use. Moreover, new methodologies to determine the bactericidal activity of these compounds have been proposed. This study describes the use of bioluminescent strains of Mycobacterium tuberculosis H37Rv - ATCC 27974 and Mycobacterium tuberculosis Erdman ATCC 35801 both containing the plasmid pSMT1 constructed with luxA and luxB genes from Vibrio harveyi in a screening to evaluate the antimycobacterial activities of anti-TB agents. Standardization of the technique was performed using isoniazid and rifampicin, as drugs standard. The results of Minimal Inhibitory Concentration (MIC) were 0.03 μg/mL and 0.03 μg/mL, respectively. These values were totally compatible with those obtained by Microplate Alamar Blue Assay (MABA). Standardization of bioluminescence measurements of intracellular antimycobacterial activity was performed using the J774 murine...(Complete abstract, click electronic access below) / Doutor

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

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第15396号 / 工博第3275号 / 新制||工||1493(附属図書館) / 27874 / 京都大学大学院工学研究科分子工学専攻 / (主査)教授 榊 茂好, 教授 白川 昌宏, 教授 北川 進 / 学位規則第4条第1項該当

Entre centenas de espécies brasileiras de pirilampos, o Pyrearinus termitilluminans é o único cujo ciclo de vida se passa integralmente nos cupinzeiros luminosos localizados no Brasil central claramente observados durante a estação das chuvas. A emissão de luz nos elaterídeos tem a função de atração de presas para a alimentação. A bioluminescência desses animais desaparece nos meses de seca juntamente com a nuvem de presas aladas. Assim, o metabolismo de trealose aqui estudado poderia prover informações sobre da capacidade da larva de sobreviver em ambientes edáficos. As concentrações de trealose e glicose são determinadas nos extratos de larvas com um sistema DIONEX® de cromatografia iônica. A trealase foi medida com 17 mM de trealose. O glicogênio foi estimado com uma amiloglicosidase. Paralelamente o estresse oxidativo associado com a restrição de água foi monitorado através da determinação do TBARS, juntamente com a catalase, glutationa peroxidase e glutationa redutase. Nas larvas submetidas ao ensaio na câmara climática (25% de umidade) a trealase (159,69±10,95 mU/animal) estava 80 vezes mais alta do que a do grupo controle (2,02±1,41mU/animal). As larvas sob estresse apresentaram distintos níveis de trealose e glicose (29,85±3,20 µmol/animal e 18,27±0,82 µmol/animal, respectivamente) quando comparadas com o grupo controle (64,61±1,54 µmol/animal e 2,16±0,11 µmol/animal, respectivamente). A elevação dos níveis das enzimas antioxidantes (4, 4,3 e 2,4 vezes respectivamente) com níveis invariáveis de TBARS apontam para a ação antioxidante contra a produção elevada de espécies reativas de oxigênio. Os insetos submetidos à restrição hídrica perderam aproximadamente 35% do peso. O aumento da atividade da trealase, com concomitante decréscimo dos níveis de trealose, sugerem que esse açúcar poderia ser usado como fonte hídrica. Entretanto, os níveis de glicose, 10 vezes mais elevados no grupo sob estresse poderia ser usado na via de biossíntese de trealose uma vez que os níveis de glicogênio estão diminuídos. As mudanças no metabolismo de trealose e o desbalanço no equilíbrio redox, poderiam fornecer importantes informações fisiológicas desses insetos sob estresse hídrico. / The life cycle of Pyrearinus termitilluminans (Coleoptera: Elateridae) takes place totally into the so-called luminous termite mounds located in Central Brazil, which are clearly observed during the rainy season. Light emission by the elaterid larvae acts like a trap attracting flying insects. The bioluminescence disappears in the dry months together with the food supply. The trehalose metabolism study described here could provide information about larva capacity to survive throughout hard climatic changes. Trehalose and glucose concentrations are determined in the larva extracts with a DIONEX® ion chromatography system. The trehalase activity was measured with 17 mM trehalose. The glycogen level was estimated with amyloglucosidase. In parallel oxidative stress associated to water deprivation was evaluated through determination of TBARS and the catalase, glutathione peroxidase and glutathione reductase activities. In larvae submitted to dryness in a growth chamber (25% humidity) we have found 80-fold higher trehalase activity (159.69±10.95 mU/animal) than in the control group raised under room conditions (2.02±1.41 mU/animal). Stressed larvae showed distinct trehalose and glucose contents (29.85±3.20 µmol/animal and 18.27±0.82 µmol/animal, respectively) when compared with the control group(64.61±1.54 µmol/animal and 2.16±0.11 µmol/animal, respectively), whereas the glycogen level was lower (11.53±2.01 mg/animal and 28.26±2.31 mg/animal, respectively). Elevation of the antioxidant enzyme levels (4-fold, 4.3-fold and 2.4-fold respectively) with maintenance of TBARS pointed to depletion to exacerbated production of reactive oxygen species. Insects submitted to water restriction lost about 35% wet weight. The observed increase of trehalase activity and concomitant decrease of trehalose level suggest that trehalose could be used as a metabolic water source. Moreover, the 10-fold higher glucose level in the stressed group could be used by the trehalose biosynthetic pathway as the glycogen level decreases in parallel. The trehalose metabolic and redox balance changes described here may shed light on the yet unknown physiological mechanisms of larval elaterid adaptation to water stress.

Anglerfishes are easily one of the most popular deep-sea creatures due to their menacing appearance, extreme sexual dimorphism, parasitic mating approach, and eye catching bioluminescent lure. Unlike most bioluminescent fishes, which intrinsically generate light, female anglerfishes belonging to nine of the 11 families within the suborder Ceratioidei (deep-sea anglerfishes) have developed a symbiotic relationship with bioluminescent bacteria that are housed within the light organs. Previous molecular work had identified symbionts from two anglerfish species as novel and possibly unculturable taxa (Haygood et al., 1992), but nothing more has been revealed about the bioluminecent symbionts of ceratioids. As part of the Gulf of Mexico Research Initiative-funded DEEPEND project (Deependconsortium.org), the objective of this study is to characterize the escal microbiome of deep-sea anglerfishes and identify potential-symbiont taxa. A total of 36 anglerfish specimens were collected on DEEPEND cruises DP01 through DP04. These specimens consist of adult and larval individuals belonging to six of the families with the suborder Ceratioidei: Ceratiidae (n=22), Oneirodidae (n=7), Linophrynidae (n=3), Melanocetidae (n=2), Centrophrynidae (n=1), Melanocetidae (n=2), Gigantactinidae (n=1). DNA was extracted from esca, skin, fin, gill, gut, and caruncle tissues, as well as seawater. High-throughput sequencing of the 16S rRNA hypervariable V4 region was carried out using the Illumina MiSeq. Sequencing revealed five potential bioluminescent-symbiont taxa (OTU IDs: 9129, 9131, 160210, 523223, and 939811), which had the greatest relative abundance (25.2% - 98.7%) within 12 of 21 adult specimens. These taxa belong to the family Vibrionaceae and were found at greater than 10% relative abundance in the escal samples of adult anglerfishes belonging to the Ceratiidae and Melanocetidae families, but they were not found in high abundance in larval individuals of the same families. Sequencing of larval samples revealed five potential bioluminescent-symbiont taxa (OTU IDs: 136178, 176420, 523223, 837366, 939811) which were of greatest relative abundance (8.1%-67.1%) within nine of 13 specimens. Also members of the family Vibrionaceae, these taxa were found in high abundance in larval anglerfishes belonging to the Oneirodidae, Linophrynidae, Gigantactinidae, and Ceratiidae families. This study is the first to to examine the bioluminescent symbionts from seven different ceratioid families.

L-arginine is one of the most versatile amino acids due to the fact that it serves as a precursor for many molecules which have important roles in bodily functions including mammalian reproduction. The current studies sought to further examine the role that L-arginine has in mammalian reproduction utilizing both in vivo and in vitro approaches. In the first study, a novel bioluminescent murine pregnancy model was developed to monitor VEGFR2 transcription activity non-invasively in the fetoplacental unit. Secondly, the effect that dietary L-arginine supplementation has during mouse gestation was examined. L-arginine supplementation increased weight gain during the latter third of gestation, total litter size, number of implantation sites, and litter birth weight. Additionally, L-arginine supplementation increased VEGFR2 transcription activity in the fetoplacental unit which may create a more favorable environment for fetal survival. Moreover, the increased number of implantation sites observed suggests an effect of L-arginine at the level of the endometrium. To this end, the effect that L-arginine has on apoptosis and cell proliferation in an established endometrial cell line was examined. The addition of L-arginine at physiological (200 micromolar) and supra-physiological (800 micromolar) concentrations increased cell proliferation , and this effect was achieved through biosynthesis of polyamines and nitric oxide. L-arginine also decreased the proportion of cells that were experiencing mitochondrial mediated apoptosis, and it was observed that this decrease in mitochondrial mediated apoptosis was concurrent with increased phosphorylation of BAD protein, which induces apoptosis when not phosphorylated. The final study examined the ability of porcine uterine epithelial (PUE) cells to synthesize L-arginine from L-citrulline. L-citrulline was able to support PUE cell proliferation in the absence of L-arginine. Additionally, ASS-1 and ASL, L-arginine synthesizing enzymes, were expressed in PUE cells and were regulated by the presence of L-arginine and L-citrulline, respectively. This data would support the hypothesis that PUE cells may be able to convert L-citrulline to L-arginine. Together, the current findings along with the plethora of relevant literature provide further evidence for the role of L-arginine in mammalian reproduction and allow for new questions to be investigated regarding this particular amino acid’s role in mammalian reproduction.

The porcine antral follicle, which consists of an oocyte and surrounding follicular components, including theca, granulosa, and cumulus cells and follicular fluid, is an essential microenvironment for oocyte development and maturation. Investigating cellular and molecular events in the context of the whole follicle will aid in our understanding of interactions between the oocyte and the follicular components. The objective of this dissertation was to develop a novel bioluminescent imaging model to visualize and measure cellular and molecular events in living intact ovarian follicles in vitro. Bioluminescence imaging was employed to facilitate noninvasive, dynamic, and real-time transgene analysis in living intact follicles. The time courses of luciferase-luciferin reactions, effective plasmid DNA and D-luciferin doses and their combinations were determined as the first step toward developing a new real-time bioluminescence imaging model. In addition, the efficient nonviral gene delivery methods: cationic lipid mediated gene transfer (chemical) and electroporation (physical) for the living intact follicles were determined. For the cationic lipid mediated gene transfer method, the 1:3 DNA lipid ratio was optimal. It was also found that the optimal condition of electroporation (4 electric pulses with 100 ms duration at field strength of 100 V/cm) resulted in 15 times higher luciferase activity and increased granulosa cell viability over the cationic lipid mediated gene transfer method. Moreover, increased granulosa cell viability, increased follicular fluid progesterone content, and oocytes with expanded cumulus cells were observed in intact follicles transfected by electroporation at a field strength of 100 V/cm. Finally, bioluminescence imaging was applied to quantify functional and ligand-activated estrogen receptor (ER) activity within living intact follicles. The functional ERs were differentially activated during the different stages of the estrous cycle in the mature sow; the levels of functional ER activity in cultured granulosa cells and intact follicles in vitro were increased from late luteal phase to early follicular phase and then significantly decreased at late follicular phase. The methodology developed herein can be applicable to further our understanding of oocyte and follicle development and oocyte maturation.

Virulent Aeromonas hydrophila (vAh) is an important emerging bacterial pathogen causing motile Aeromonas septicemia (MAS) in farmed catfish. Understanding the pathogenicity of the disease is essential for the development of preventive measures. In this study, we aimed to develop a bioluminescent virulent A. hydrophila (BvAh) strain to understand the pathogen-host interactions during infection. To achieve this, a new bioluminescence expression plasmid, pAKgfplux3, was constructed and mobilized to vAh. Catfish were challenged with BvAh using immersion, injection, and adipose fin clip procedures, and bioluminescence signal was tracked in live catfish during infection. We developed a novel BvAh strain for the first time, conducted imaging of BvAh in live fish, detected infection routes and attachment sites of the pathogen, and determined target organs, which provided new insights on the pathogenesis of vAh. MAS progressed better in fish when protection of skin was bypassed. Abraded skin seems to provide a potential portal of entry during vAh infection.

Les thérapies géniques et cellulaires ouvrent de nouvelles perspectives pour le traitement de pathologies très diverses. Cependant, l’adressage à un organe cible, le contrôle non invasif de l’expression d’un transgène et le suivi par imagerie constituent encore des défis majeurs pour le développement de ces approches thérapeutiques. L’utilisation d’un vecteur cellulaire modifié génétiquement pour exprimer un transgène spécifique semble une solution prometteuse mais il convient aussi de restreindre l’expression du transgène à la région cible et de contrôler l’expression génique dans le temps. Pour le contrôle spatio temporel de l’expression, nous proposons une approche in vivo originale qui consiste à placer le gène thérapeutique sous le contrôle d’un promoteur thermo-inductible et à contrôler l’expression par une hyperthermie localisée induite à l’aide d’Ultrasons Focalisés guidés par Imagerie de Résonance Magnétique (IRM) de température (MRgHIFU).La stratégie expérimentale consiste à générer une souris chimère par greffe de moelle osseuse. La souris donneuse est une souris transgénique qui exprime le gène rapporteur bioluminescent de la luciférase Firefly (lucF) et le gène rapporteur fluorescent de la protéine mPlum sous le contrôle du promoteur thermosensible Hsp70a1b. La souris receveuse est une souris congénique prétraitée au Busilvex® pour créer une aplasie médullaire. Deux mois après, le pourcentage de greffe est déterminé par cytométrie de flux puis une tumeur sous-cutanée est induite sur la patte arrière gauche des souris par injection sous-cutanée de cellules tumorales (Carcinoma Mouse Tumor: CMT-93). Pendant la période de croissance tumorale (1 mois) les processus physiologiques conduisent au recrutement des cellules sanguines circulantes dans la zone péri-tumorale sans expression des transgènes. L’activation du promoteur Hsp70 est alors réalisée en créant une hyperthermie locale par MRgHIFU. L’IRM permet d’obtenir des cartes thermiques utilisées pour asservir un générateur d’ultrasons et pour contrôler le chauffage (45°C, 8 min, +/- 1°C). L’expression de la lucF est détectée in vivo 6 H après chauffage par imagerie de bioluminescence (BLI) alors que l’expression de la protéine mPlum est révélée 30 H après chauffage par imagerie de fluorescence par réflectance (FRI). A la suite de l’imagerie, les souris sont sacrifiées et une analyse histologique de la tumeur et des tissus périphériques est réalisée grâce à l’expression de la protéine mPlum. Les gènes rapporteurs lucF et mPlum sont exprimés après thermo-induction principalement par les macrophages de la souris chimère et l’expression intervient, comme attendu, dans les zones péri-tumorales chauffées. Parfois, l’expression des gènes rapporteurs est détectée dans des zones non ciblées et résulte de l’expression par les progéniteurs myéloïdes situés au niveau de la moelle osseuse en réponse à l’échauffement des os par les ultrasons.Nous avons ainsi pu démontrer in vivo la faisabilité de l’approche thérapeutique proposée et mis en évidence certaines limites de la procédure expérimentale essentiellement inhérentes à la taille du modèle animal et aux méthodes physiques utilisées pour l’activation in vivo du transgène.Nous proposons également une stratégie in vivo afin d’évaluer les performances de la tomographie moléculaire de fluorescence 3D pour suivre in vivo, le devenir d’une nano-émulsions fluorescentes (LNP) chez des souris porteuses de tumeurs intracrâniennes. / Gene and cell therapies offer new perspectives for the treatment of various pathologies. However, the efficiency of the delivery method to targeted organs, the control of gene expression and imaging gene expression remain major challenges for the development of these therapeutic approaches. The combination of a cell vector and a gene modification of this vector to express a specific transgene seems a promising strategy. But even addressed, it remains very difficult to restrict and control gene expression to the target region in time and space. That is why we investigate an in vivo strategy to deliver transgenes under the transcriptional control of a thermosensitive promoter and induce “on demand” expression by local hyperthermia using Magnetic Resonance guided High-Intensity Focused Ultrasound (MRgHIFU).This strategy consisted of engraftment of transgenic bone marrow cells (BMC) to create a chimera. The donor mouse was a transgenic mouse expressing the firefly luciferase (lucF) and the fluorescent reporter of the mPlum protein under the transcriptional control of the thermosensitve promoter Hsp70a1b. The mouse receiver was a congenic mouse pre-treated with Busilvex® to induce medular aplasia. Engraftment efficiency was measured 2 months later by flux cytometry, after which a tumor was implanted on the left leg of mice by subcutaneous injection carcinoma mouse tumor-93 cells. During tumor growth (1 month) circulating blood cells accumulated in and around the tumor as part of an inflammatory process without transgene expression. Local activation of the thermosensitive promoter Hsp70 was induced by hyperthermia using MRgHIFU. MRI temperature maps were used to provide feedback to an ultrasound generator and to control heating (45°C, 8 min, +/-1°C). The expression of lucF was detected by in vivo imaging of bioluminescence (BLI) 6 H after heating while mPlum expression was revealed by fluorescence that appeared 30 H post heating. After imaging, mice were sacrified and immunohistochemical analysis on tumors and tissues performed to identify mPlum expressing cells. Local heating of tumors induced expression of transgenes placed under control of the Hspa1b promoter in bone marrow-derived cells. Most of these cells remained in the vicinity of or within the tumor as both luciferase activity 6 H and mPlum fluorescence 30 H post-heating were concentrated at the tumor site. LucF and mPlum expression were caracterized in vivo and in vitro. In some cases, unexpected signal appear in non-heated specific areas corresponding to bone. Histology revealed the presence of mPlum in tumor macrophages and bone marrow myeloid progenitors. Having demonstrated the feasibility of this approach, our results also reveal some limits of these approaches that are inherent to the size of the animal model and the physical methods for in vivo activation of the transgene. We now propose an in vivo strategy to evaluate the performances of 3D fluorescent molecular tomography to follow in vivo the presence of intracerebral tumors in nude mice by using a fluorescent nano-emulsion (LNP).