L’objectif général du travail de thèse a donc été d’analyser le rôle respectif du volume tumoral et des paramètres pharmacocinétiques dans la réponse au rituximab en utilisant des moyens d’imagerie adaptés aux modèles murins et à la cancérologie. Dans une première partie nous avons utilisé une lignée lymphomateuse T (El4) syngénique de souris C57Bl6J, transduite par le CD20 humain et transfectée avec le gène de la luciférase. Dans une seconde partie une seule injection de rituximab a été réalisée et la concentration a été évaluée par une méthode ELISA. L’analyse des concentrations au cours du temps nous a permis de montrer une très grande variabilité d’exposition au rituximab semblable à l’observation faite chez l’homme. Nous avons pu modéliser les concentrations de rituximab et la progression des foyers tumoraux par la construction d’un modèle concentration/effet nous permettant de démontrer l’existence d’une relation entre l’efficacité du rituximab et le volume tumoral. Enfin dans un troisième volet nous avons utilisé le modèle cellulaire afin d’évaluer in vitro l’usage de particules d’oxydes de gadolinium ou de bore. / The overall objective of this work of thesis was to analyze the respective role of tumor burden and pharmacokinetic parameters in the response to the rituximab by using systems of imagery adapted to murine models and to cancerology. In a first part of development of the model we used a T lymphoma cell line (El4) syngenic of mouse C57Bl6J, transduted by the human CD20 and transfected with luciferase gene. In a second part, one injection of rituximab was carried out and the circulating concentration was evaluated by an ELISA method. The analysis of the rituximab concentrations in the course of time enabled us to show a very important variability of exposure to the rituximab similar to the observation made for the human. We realized a model of the rituximab concentrations and of the tumors evolution by the construction of a concentration/effect model allowing us to show the existence of a relation between the effectiveness of the rituximab and tumor burden before treatment. Finally in a third part we used the cellular model El4-huCD20-Luc in order to evaluate in vitro the use of gadolinium oxide particles or boron and gadolinium oxide particles.

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.

Este tese descreve métodos para a purificação e determinação da massa molecular do precursor da luciferina fúngica, a partir dos corpos de frutificação do fungo bioluminescente Neonothopanus gardneri. A molécula em questão é o substrato da primeira etapa da reação enzimática responsável pela emissão de luz em fungos, fenômeno conhecido como bioluminescência. Ao longo do projeto, foi otimizado um ensaio analítico qualitativo para detecção do precursor da luciferina em solução, e também foram desenvolvidos métodos de extração e purificação da molécula de interesse, levando sempre em consideração a baixa quantidade da molécula disponível in vivo e a susceptibilidade de degradação da molécula quando exposta a oxigênio, luz e pH extremos. Após estabelecer diversas condições cromatográficas para a purificação do precursor da luciferina, foi possível correlacionar uma molécula de massa molecular 246,05 u com uma atividade positiva no ensaio analítico qualitativo para detecção da luciferina em solução. Posteriormente, foi descoberto por um grupo russo que o precursor da luciferina tinha massa molecular 246,05 u e fórmula molecular C13H10O5. Também foram realizados experimentos a fim de corroborar a estrutura molecular do produto da reação de emissão de luz em fungos, assim como determinar o seu mecanismo de formação. / This thesis describes methods for purification and determination of molecular mass of fungal luciferin precursor from fruiting bodies of bioluminescent fungus Neonothopanus gardneri. The molecule it is the substrate of enzymatic reaction first step responsible for light emission in fungi, phenomenon known as bioluminescence. During PhD, it was optimized a qualitative assay in order to detect the luciferin precursor in solutions and also were developed methods for extraction and purification of the molecule of interest, always considering the low amount of molecule available in vivo and the susceptibility of molecule degradation when exposed to oxygen, light and extremes pH\'s. After establishing several chromatographic conditions for purification of luciferin precursor, it was possible to correlate a molecule with molecular mass of 246.05 u with a positive activity in the qualitative analytical assay. Later, a Russian group describe that the precursor of luciferin had molecular mass 246.05 u and molecular formula C13H10O5. Experiments were also performed in order to corroborate the molecular structure of the reaction product of light emission in fungi as well as determine its formation mechanism.

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

Este tese descreve métodos para a purificação e determinação da massa molecular do precursor da luciferina fúngica, a partir dos corpos de frutificação do fungo bioluminescente Neonothopanus gardneri. A molécula em questão é o substrato da primeira etapa da reação enzimática responsável pela emissão de luz em fungos, fenômeno conhecido como bioluminescência. Ao longo do projeto, foi otimizado um ensaio analítico qualitativo para detecção do precursor da luciferina em solução, e também foram desenvolvidos métodos de extração e purificação da molécula de interesse, levando sempre em consideração a baixa quantidade da molécula disponível in vivo e a susceptibilidade de degradação da molécula quando exposta a oxigênio, luz e pH extremos. Após estabelecer diversas condições cromatográficas para a purificação do precursor da luciferina, foi possível correlacionar uma molécula de massa molecular 246,05 u com uma atividade positiva no ensaio analítico qualitativo para detecção da luciferina em solução. Posteriormente, foi descoberto por um grupo russo que o precursor da luciferina tinha massa molecular 246,05 u e fórmula molecular C13H10O5. Também foram realizados experimentos a fim de corroborar a estrutura molecular do produto da reação de emissão de luz em fungos, assim como determinar o seu mecanismo de formação. / This thesis describes methods for purification and determination of molecular mass of fungal luciferin precursor from fruiting bodies of bioluminescent fungus Neonothopanus gardneri. The molecule it is the substrate of enzymatic reaction first step responsible for light emission in fungi, phenomenon known as bioluminescence. During PhD, it was optimized a qualitative assay in order to detect the luciferin precursor in solutions and also were developed methods for extraction and purification of the molecule of interest, always considering the low amount of molecule available in vivo and the susceptibility of molecule degradation when exposed to oxygen, light and extremes pH\'s. After establishing several chromatographic conditions for purification of luciferin precursor, it was possible to correlate a molecule with molecular mass of 246.05 u with a positive activity in the qualitative analytical assay. Later, a Russian group describe that the precursor of luciferin had molecular mass 246.05 u and molecular formula C13H10O5. Experiments were also performed in order to corroborate the molecular structure of the reaction product of light emission in fungi as well as determine its formation mechanism.

The Earth’s oblique rotation results in changes in light and temperature across the day and time of year. Living organisms evolved rhythmic behaviours to anticipate these changes and execute appropriate responses at particular times. The current paradigm for the biological clocks in several branches of life is an underlying biochemical oscillator mainly composed by a network of repressive transcription factors. The slow decay in their activity is fundamental for generating anticipatory dynamics. Interestingly, these dynamics can be well appreciated when the biological system is left under constant environmental conditions, where oscillation of several physiological readouts persists with a period close to 24 hours, hence the term “circadian clocks”, circa=around dian=day. In plants the model species Arabidopsis thaliana has served as an invaluable tool for analysing the genetics, biochemical, developmental, and physiological effects of the oscillator. Many of these experimental results have been integrated in mechanistic and mathematical theories for the circadian oscillator. These models predict the timing of gene expression and protein presence in several genetic backgrounds and photoperiodic conditions. The aim of this work is the introduction of a correct mass scale for both the RNA transcript and protein variables of the clock models. The new mass scale is first introduced using published RNA data in absolute units, from qRT-PCR. This required reinterpreting several assumptions of an established clock model (P2011), resulting in an updated version named U2017. I evaluate the performance of the U2017 model in using data in absolute mass units, for the first time for this clock system. Introducing absolute units for the protein variables takes place by generating hypothetical protein data from the existing qRT-PCR data and comparing a data-driven model with western blot data from the literature. I explore the consequences of these predicted protein numbers for the model’s dynamics. The process required a meta-analysis of plant parameter values and genomic information, to interpret the biological relevance of the updated protein parameters. The predicted protein amounts justify, for example, the revised treatment of the Evening Complex in the U2017 model, compared to P2011. The difficulties of introducing absolute units for the protein components are discussed and components for experimental quantification are proposed. Validating the protein predictions required a new methodology for absolute quantification. The methodology is based on translational fusions with a luciferase reporter than has been little used in plants, NanoLUC. Firstly, the characterisation of NanoLUC as a new circadian reporter was explored using the clock gene BOA. The results show that this new system is a robust, sensitive and automatable approach for addressing quantitative biology questions. I selected five clock proteins CCA1, LHY, PRR7, TOC1 and LUX for absolute quantification using the new NanoLUC methodology. Functionality of translation fusions with NanoLUC was assessed by complementation experiments. The closest complementing line for each gene was selected to generate protein time series data. Absolute protein quantities were determined by generation of calibration curves using a recombinant NanoLUC standard. The developed methodology allows absolute quantification comparable to the calibrated qRT-PCR data. These experimental results test the predicted protein amounts and represent a technical resource to understand protein dynamics of Arabidopsis’ circadian oscillator quantitatively. The new experimental, meta-analysis and modelling results in absolute units allows future researchers to incorporate further, quantitative biochemical data.

Made available in DSpace on 2014-06-11T19:31:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2003Bitstream added on 2014-06-13T20:01:22Z : No. of bitstreams: 1 sato_dn_dr_araiq.pdf: 232098 bytes, checksum: fef9938402c38520312a4f3268b1687d (MD5) / 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... / 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)

Graduation date: 2001

Cell-based insulin therapies can potentially improve glycemic regulation in insulin dependent diabetes patients and thus help reduce secondary complications. The long-term goal of our work is to engineer autologous insulin-secreting intestinal endocrine cells as a non-beta cell approach to alleviate donor cell shortage and immune rejection issues associated with islet transplantation. These cells have been chosen for their endogenous similarity to beta cells, but generating cell constructs with sufficient insulin secretion for therapeutic effect has proven challenging. Previous work in our lab showed that a tissue engineered pancreatic substitute (TEPS) based on an engineered insulin-secreting L cell line, GLUTag-INS, was insufficient in affecting blood glucose levels in streptozotocin-induced diabetic mice, but promising since human insulin was detected in the blood. The objective of this project was therefore to fabricate an improved TEPS based on GLUTag-INS cells and evaluate its suitability as a standalone diabetes therapy. To achieve this objective, the following specific aims were (1) to investigate gene incorporation as a strategy to enhance recombinant insulin secretion from GLUTag-INS cells; (2) to develop and characterize a TEPS in vitro based on a microcapsule system containing improved GLUTag-INS cells with bioluminescence monitoring capability; and (3) to assess therapeutic efficacy of the graft in a diabetic, immune-competent mouse model and use bioluminescence monitoring to elucidate in vivo transplant behavior. This thesis therefore reports on the progression of studies from the genetic and molecular levels for improved insulin secretion per-cell, to the tissue level for enhanced secretion per-graft, and lastly to the preclinical level for therapeutic assessment in a diabetic mouse model.

Thesis (Ph. D.)--University of California, San Diego, 1998. / Vita. Includes bibliographical references.