Despite the fact that some aspects of single studies differ, there's a generally accepted view that the whole cartilaginous viscerocranium of vertebrates is neural crest derived. By the series of isotopic transplantation experiments of presumptive neural crest on the model organism Ambystoma mexicanum I partly specify this oppinion and prove that the most ventro-caudal cartilage, the second basibranchial, is of a different origin. Furher I mention the level of the presumptive neural crest where the single parts of cartilaginous viscerocranium arise from. Moreover there is one element, the first basibranchial, which has double origin. I discuss also some other neural crest derivatives such as head and outer gills mesenchyme, the trabeculae cranii, part of the cartilaginous otic capsule or the connective tissue in the head. I have performed 179 transplantations between transgenic and normal axolotl embryos. My final analysis is composed of 65 embryos of stage 40 - 42 and 7 larvae of lenght of 15 - 17 mm.

No description available.

No description available.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Raymond R. R. Rowland / Mechanisms for mutations in RNA viruses include random point mutations, insertions, deletions, recombination and re-assortment. Most viruses have more than one of these mechanisms operating during their life cycle. Impact of sequence divergence is seen in the areas of evolution, epidemiology and ecology of these viruses. Immediate negative consequences of genetic diversity include failure of vaccination, resistance to anti-virals, emergence and re-emergence of novel virus isolates with increased virulence or altered tropism. To identify specific sequence features that influence recombination, a new in-vitro system was developed using an infectious cDNA clone of PRRS virus that expressed fluorescent proteins. The in-vitro experimental system involved the co-transfection of a pair of closely related PRRSV infectious clones: a fully functional non-fluorescent PRRS virus infectious clone that possessed a single mutation in a green fluorescent protein (GFP) and a second infectious clone that contained a defective fluorescent virus. The readout for successful recombination was appearance of a fully functional fluorescent virus. The model system creates the opportunity to study several aspects of recombination, including the requirement for sequence homology between viruses undergoing recombination.

Adenosine Triphosphate (ATP) can be released as a signal between cells in an autocrine and paracrine manner that binds purinergic receptors. Highly conserved, purinergic receptors expressed on the cell surface of neurons and astrocytes are capable of being activated across eight orders of magnitude from hundreds of nanomolar ATP to millimolar. Genetically encoded fluorescent protein biosensors have been used to detect ATP outside the cell, but a high affinity extracellular ATP sensor is required to study the ATP signaling dynamics from nanomolar to micromolar magnitudes. Previously, our lab developed a first generation sensor of extracellular ATP called ECATS1 (Conley et al.). To develop an improved sensor, we caried out site-directed mutagenesis of the sensor's ATP binding site and identified a mutant that exhibited a 4-fold increase in ATP binding affinity in solution. We then optimized the membrane-tethering of the sensor to achieve the 4-fold increase in extracellular ATP binding affinity when measured on live cell.s This second-generation sensor was dubbed ECATS2. As a proof-of-concept application, we sought to detect ATP release from cells using <i>in vitro</i> models of edema. We subjected HEK293A cells to hypo-osmotic shock (HOS), revealing ATP release at micromolar levels. Then we tested HOS in cultured cortical astrocytes, also revealing micromolar ATP release. However, when we tested neuron-astrocyte co-cultures, we no longer observed ATP release in response to HOS. Interestingly, this implies that co-culture either entirely prevented ATP release from astrocytes or dampened it into the nanomolar range below the limit of ECATS2 detection. Thus, we have validated the development of a higher affinity, second-generation sensor and used it to discover that ATP release from astrocytes after HOS can be affected by the presence of neurons. <br>

Devido ao número crescente de surtos de infecção hospitalar, torna-se proeminente o estabelecimento de um programa de sanitização que liste os agentes químicos a serem empregados e o modo de aplicação mais efetivo. Processos de desinfecção também são relevantes em sistemas de tratamento de água (em indústrias farmacêuticas e centros de saúde) para que a qualidade da água seja assegurada e atenda os parâmetros estabelecidos, evitando proliferação microbiana. Validação da eficácia de descontaminação é uma tarefa ao mesmo tempo importante e desafiadora. Indicadores biológicos são sistemas ou moléculas que detectam atividade biológica, permitindo a validação de processos de descontaminação ou desinfecção. O indicador biológico pode ser uma suspensão de microrganismos específicos (sistema biológico) com resistência definida a um determinado processo de descontaminação. Enzimas e proteínas também têm sido empregadas como indicadores biológicos para avaliar a eficácia de processos industriais. A proteína verde fluorescente (GFP) tem sido sugerida como potencial indicador biológico para tratamentos de desinfecção, devido facilidade de sua detecção por espectrofluorimetria ou por inspeção visual. Para estudar e comparar o comportamento dos microrganismos selecionados e da GFP foram realizados ensaios de concentração inibitória mínima (CIM) e tempo de redução decimal (valor D). A CIM capaz de reduzir o bioburden inicial (>8log10) foi: 59 - 156 mg/L- 3250 mg/mL de glutaraldeído, 39 - 246 mg/L de formaldeído, 43750 - 87500 mg/L de álcool etílico 1250 - 6250 mg/L de polivinilpirrolidona iodo, 150 - 4491 mg/L de compostos liberadores de cloro, 469 -2500 mg/L de peróxido de hidrogênio e 2310 - 18500 mg/L de ácido peracético. A. calcoaceticus apresentou resistência à maioria dos agentes químicos testados, seguido de E. cloacae e S. marcescens. No sistema de purificação de água os resultados para Pseudomonas aeruginosa foram: (i) 0,5% de ácido cítrico, D = 3,8 min; (ii) 0,5% de ácido clorídrico, D = 6,9 min; (iii) 70% álcool etílico, D = 9,7 min; (iv) 0,5% bissulfito de sódio, D = 5,3 min; (v) 0,4% de hidróxido de sódio, D = 14,2 min; (vi) 0,5% de hipoclorito de sódio, D = 7,9 min; (vii) mistura de peróxido de hidrogênio (2,2%) e ácido peracético (0,45%), D = 5,5 min. GFP testada frente a soluções cloradas (com diferentes valores de pH e concentração) resultou em diminuição da fluorescência, sendo mais evidente em concentrações de cloro maiores que 150 ppm, com valores D entre 1,3 - 1,7 min. Em soluções de cloro em tampão fosfato (pH=7,15±0,08), a proteína manteve sua estrutura em contato com soluções 52-94 ppm de cloro. Frente a 110 ppm de cloro a estabilidade da proteína foi reduzida 10 vezes. A proteína GFP se mostrou um marcador fluorescente apropriado para monitorar eficácia de desinfecção. Para ser empregada como indicador biológico a proteína deve ser purificada através de um método de fácil ampliação de escala e com boa relação custo benefício, com este intuito o sistema micelar de duas fases aquosas foi estudado, e a proteína GFP foi parcialmente recuperada do homogeneizado celular de E. coli, outros contaminantes presentes no meio foram removidos na mesma etapa. A demonstração de que é possível se extrair uma biomolécula alvo utilizando ligantes de afinidade representa um passo importante no desenvolvimento de um método eficaz de separação que poderá ser utilizado na purificação de outras biomoléculas. / Due to the growing number of outbreaks of infection in hospital and nurseries, it becomes essential to set up a sanitation program that indicates that the appropriate chemical agent was chosen for application in the most effective way. Disinfection processes are also relevant in water purification systems (in pharmaceutical industries and in health environments) to assure water quality, meeting ionic and organic chemical standards, and avoiding microbial proliferation. Validating the effectiveness of decontamination and disinfection is an important and often challenging task. A biological indicator is a system or a molecule that enables the detection of biological activity, and as such, permits the validation of decontamination or disinfection treatments. The biological indicator can be a specific microorganism suspension (microbiological test system) with a defined resistance to a particular decontamination treatment. Enzymes and proteins have also been used as biological indicators to evaluate the immediate efficacy of industrial procedures, such as blanching, pasteurization, and disinfection treatments, as well as to monitor the satisfactory preservation of a product subjected to disinfection or sterilization. Green fluorescent protein (GFP) has been proposed as an ideal choice for a protein-based biological indicator for use in the validation of decontamination or disinfection treatments. In order to study and compare microorganism (in hospital infections outbreaks and isolated from the water purification system) and protein behavior, microorganisms were submitted to minimal inhibitory concentration (CIM) and decimal reduction time determination (D value). The CIM intervals, which reduced bacteria populations over 8log10, were: 59 to 156 mg/L of quaternarium ammonium compounds (QACs); 63 to 10000 mg/L of chlorhexidine; 1375 to 3250 mg/L of glutaraldehyde; 39 to 246 mg/L of formaldehyde; 43750 to 87500 mg/L of ethanol; 1250 to 6250 mg/L of iodine in polyvinyl-pyrolidone complexes, 150 to 4491 mg/L of chlorine-releasing-agents (CRAs); 469 to 2500 mg/L of hydrogen peroxide; and, 2310 to 18500 mg/L of peracetic acid. Chlorhexidine showed non inhibitory activity over germinating spores. A. calcoaceticus showed resistance to the majority of the agents tested, followed by E. cloacae and S. marcescens. In the water purification system the results were for P. aeruginosa into: (i) 0.5% citric acid, D = 3.8 min; (ii) 0.5% hydrochloric acid, D = 6.9 min; (iii) 70% ethanol, D = 9.7 min; (iv) 0.5% sodium bisulfite, D = 5.3 min; (v) 0.4% sodium hydroxide, D = 14.2 min; (vi) 0.5% sodium hypochlorite, D = 7.9 min; (vii) mixture of hydrogen peroxide (2.2%) plus peracetic acid (0.45%), D = 5.5 min. GFP was challenged with chlorine solutions (different pH and chlorine concentrations) and its fluorescence decreased abruptly on contact with chlorine in concentrations greater than 150 ppm, with D-values between 1.3 min (147 ppm chlorine) and 1.7 min (183 ppm chlorine). In phosphate buffered chlorine solutions (pH=7.15±0.08), GFP maintained its structure between 52-94 ppm of chlorine, but protein stability decreased 10-fold when exposed to 110 ppm chlorine. GFP performed as a suitable fluorescent marker for monitoring disinfection effectiveness. To be used as a biological indicator GFP has to be purified through a potentially scalable and cost-effective way to purify the recombinant protein, produced by E. coli. The method studied was two-phase aqueous micellar system, and GFP was partially recovered from a clarified E. coli cell lysate. Other contaminating proteins were simultaneously removed. The demonstration of proof-ofprinciple of the direct affinity-enhanced extraction of CBM9-GFP from the cell lysate represents an important first step towards developing a cost-effective separation method for GFP, and more generally, for other proteins of interest.

Devido ao número crescente de surtos de infecção hospitalar, torna-se proeminente o estabelecimento de um programa de sanitização que liste os agentes químicos a serem empregados e o modo de aplicação mais efetivo. Processos de desinfecção também são relevantes em sistemas de tratamento de água (em indústrias farmacêuticas e centros de saúde) para que a qualidade da água seja assegurada e atenda os parâmetros estabelecidos, evitando proliferação microbiana. Validação da eficácia de descontaminação é uma tarefa ao mesmo tempo importante e desafiadora. Indicadores biológicos são sistemas ou moléculas que detectam atividade biológica, permitindo a validação de processos de descontaminação ou desinfecção. O indicador biológico pode ser uma suspensão de microrganismos específicos (sistema biológico) com resistência definida a um determinado processo de descontaminação. Enzimas e proteínas também têm sido empregadas como indicadores biológicos para avaliar a eficácia de processos industriais. A proteína verde fluorescente (GFP) tem sido sugerida como potencial indicador biológico para tratamentos de desinfecção, devido facilidade de sua detecção por espectrofluorimetria ou por inspeção visual. Para estudar e comparar o comportamento dos microrganismos selecionados e da GFP foram realizados ensaios de concentração inibitória mínima (CIM) e tempo de redução decimal (valor D). A CIM capaz de reduzir o bioburden inicial (>8log10) foi: 59 - 156 mg/L- 3250 mg/mL de glutaraldeído, 39 - 246 mg/L de formaldeído, 43750 - 87500 mg/L de álcool etílico 1250 - 6250 mg/L de polivinilpirrolidona iodo, 150 - 4491 mg/L de compostos liberadores de cloro, 469 -2500 mg/L de peróxido de hidrogênio e 2310 - 18500 mg/L de ácido peracético. A. calcoaceticus apresentou resistência à maioria dos agentes químicos testados, seguido de E. cloacae e S. marcescens. No sistema de purificação de água os resultados para Pseudomonas aeruginosa foram: (i) 0,5% de ácido cítrico, D = 3,8 min; (ii) 0,5% de ácido clorídrico, D = 6,9 min; (iii) 70% álcool etílico, D = 9,7 min; (iv) 0,5% bissulfito de sódio, D = 5,3 min; (v) 0,4% de hidróxido de sódio, D = 14,2 min; (vi) 0,5% de hipoclorito de sódio, D = 7,9 min; (vii) mistura de peróxido de hidrogênio (2,2%) e ácido peracético (0,45%), D = 5,5 min. GFP testada frente a soluções cloradas (com diferentes valores de pH e concentração) resultou em diminuição da fluorescência, sendo mais evidente em concentrações de cloro maiores que 150 ppm, com valores D entre 1,3 - 1,7 min. Em soluções de cloro em tampão fosfato (pH=7,15±0,08), a proteína manteve sua estrutura em contato com soluções 52-94 ppm de cloro. Frente a 110 ppm de cloro a estabilidade da proteína foi reduzida 10 vezes. A proteína GFP se mostrou um marcador fluorescente apropriado para monitorar eficácia de desinfecção. Para ser empregada como indicador biológico a proteína deve ser purificada através de um método de fácil ampliação de escala e com boa relação custo benefício, com este intuito o sistema micelar de duas fases aquosas foi estudado, e a proteína GFP foi parcialmente recuperada do homogeneizado celular de E. coli, outros contaminantes presentes no meio foram removidos na mesma etapa. A demonstração de que é possível se extrair uma biomolécula alvo utilizando ligantes de afinidade representa um passo importante no desenvolvimento de um método eficaz de separação que poderá ser utilizado na purificação de outras biomoléculas. / Due to the growing number of outbreaks of infection in hospital and nurseries, it becomes essential to set up a sanitation program that indicates that the appropriate chemical agent was chosen for application in the most effective way. Disinfection processes are also relevant in water purification systems (in pharmaceutical industries and in health environments) to assure water quality, meeting ionic and organic chemical standards, and avoiding microbial proliferation. Validating the effectiveness of decontamination and disinfection is an important and often challenging task. A biological indicator is a system or a molecule that enables the detection of biological activity, and as such, permits the validation of decontamination or disinfection treatments. The biological indicator can be a specific microorganism suspension (microbiological test system) with a defined resistance to a particular decontamination treatment. Enzymes and proteins have also been used as biological indicators to evaluate the immediate efficacy of industrial procedures, such as blanching, pasteurization, and disinfection treatments, as well as to monitor the satisfactory preservation of a product subjected to disinfection or sterilization. Green fluorescent protein (GFP) has been proposed as an ideal choice for a protein-based biological indicator for use in the validation of decontamination or disinfection treatments. In order to study and compare microorganism (in hospital infections outbreaks and isolated from the water purification system) and protein behavior, microorganisms were submitted to minimal inhibitory concentration (CIM) and decimal reduction time determination (D value). The CIM intervals, which reduced bacteria populations over 8log10, were: 59 to 156 mg/L of quaternarium ammonium compounds (QACs); 63 to 10000 mg/L of chlorhexidine; 1375 to 3250 mg/L of glutaraldehyde; 39 to 246 mg/L of formaldehyde; 43750 to 87500 mg/L of ethanol; 1250 to 6250 mg/L of iodine in polyvinyl-pyrolidone complexes, 150 to 4491 mg/L of chlorine-releasing-agents (CRAs); 469 to 2500 mg/L of hydrogen peroxide; and, 2310 to 18500 mg/L of peracetic acid. Chlorhexidine showed non inhibitory activity over germinating spores. A. calcoaceticus showed resistance to the majority of the agents tested, followed by E. cloacae and S. marcescens. In the water purification system the results were for P. aeruginosa into: (i) 0.5% citric acid, D = 3.8 min; (ii) 0.5% hydrochloric acid, D = 6.9 min; (iii) 70% ethanol, D = 9.7 min; (iv) 0.5% sodium bisulfite, D = 5.3 min; (v) 0.4% sodium hydroxide, D = 14.2 min; (vi) 0.5% sodium hypochlorite, D = 7.9 min; (vii) mixture of hydrogen peroxide (2.2%) plus peracetic acid (0.45%), D = 5.5 min. GFP was challenged with chlorine solutions (different pH and chlorine concentrations) and its fluorescence decreased abruptly on contact with chlorine in concentrations greater than 150 ppm, with D-values between 1.3 min (147 ppm chlorine) and 1.7 min (183 ppm chlorine). In phosphate buffered chlorine solutions (pH=7.15±0.08), GFP maintained its structure between 52-94 ppm of chlorine, but protein stability decreased 10-fold when exposed to 110 ppm chlorine. GFP performed as a suitable fluorescent marker for monitoring disinfection effectiveness. To be used as a biological indicator GFP has to be purified through a potentially scalable and cost-effective way to purify the recombinant protein, produced by E. coli. The method studied was two-phase aqueous micellar system, and GFP was partially recovered from a clarified E. coli cell lysate. Other contaminating proteins were simultaneously removed. The demonstration of proof-ofprinciple of the direct affinity-enhanced extraction of CBM9-GFP from the cell lysate represents an important first step towards developing a cost-effective separation method for GFP, and more generally, for other proteins of interest.

In recent years, the study of stimulated emission from and by biological systems has gained wide spread attention as a promising technology platform for novel biointegrated laser. However, the photonic properties and the associated physics of many biological laser systems are not yet fully understood and many promising resonator architectures and laser classes have not yet transitioned into the biological world. In this thesis, we investigate the fundamental photonic properties of lasers based on single biological cells and explore the potential of distributed feedback (DFB) gratings as novel biointegrated laser resonators. We show how the easy and flexible fabrication of DFB resonators helps to realize optofluidic and solid-state biological lasers. Lasing characteristics, such as tunable and single mode emission, are investigated and different applications are explored. Fourier-space emission studies on different biological lasers give insight in to the photonic dispersion relation of the system and the fundamental creation of lasing modes and their confinement in living systems. The first purely water based optofluidic DFB laser is demonstrated and novel sensing applications are suggested. This device shows low threshold lasing due to an optimized mode shape, which is achieved by a low refractive index substrate and the use of a mixed-order grating. Next, by integrating a high refractive index interlayer on a DFB resonator, a laser device incorporating the novel solid-state biological gain material green fluorescent protein (GFP) is realized. Lastly, we show how the thickness of organic polymer lasers can be reduced to its fundamental limit (< 500 nm) and the resulting membrane like laser devices can be applied to and operated on various body parts to potentially complement biometric identification.

Since the publication of the complete sequence of the human genome in 2003 there has been great interest in exploring the functions of the proteins encoded by the genes. To reveal the function of each and every protein, investigation of protein localization at the subcellular level has become a central focus in this research area, since the localization and function of a protein is closely related. The objective of the studies presented in this doctoral thesis was to systematically explore the human proteome at the subcellular level using bioimaging and to develop techniques for validation of the results obtained. A common imaging technique for protein detection is immunofluorescence (IF), where antibodies are used to target proteins in fixated cells. A fixation protocol suitable for large-scale IF studies was developed and optimized to work for a broad set of proteins. As the technique relies on antibodies, validation of their specificity to the target protein is crucial. A platform based on siRNA gene silencing in combination with IF was set-up to evaluate antibody specificity by quantitative image analysis before and after suppression of its target protein. As a proof of concept, the platform was then used for validation of 75 antibodies, proving it to be applicable for validation of antibodies in a systematic manner. Because of the fixation, there is a common concern about how well IF data reflects the in vivo subcellular distribution of proteins. To address this, 500 proteins were tagged with green fluorescent protein (GFP) and used to compare protein localization results between IF to those achieved using GFP tagged proteins in live cells. It was concluded that protein localization data from fixated cells satisfactory represented the situation in vivo and together exhibit a powerful approach for confirming localizations of yet uncharacterized proteins. Finally, a global analysis based on IF data of approximately 20 % of the human proteome was performed, providing a first overview of the subcellular landscape in three different cell lines. It was found that the intracellular distribution of proteins is complex, with many proteins occurring in several organelles. The results also confirmed the close relationship between protein function and localization, which in a way further strengthens the accuracy of the IF approach for detection of proteins at the subcellular level. / <p>QC 20121017</p> / The Human Protein Atlas

La Enhanced Cyan Fluorescent Protein (ECFP) est un variant spectral de la Green Fluorescent Protein extraite de la méduse Aequaria Victoria (GFPav). La ECFP, émettant dans le cyan, est un des donneurs les plus utilisés dans les études de transfert résonnant d'énergie d'excitation et est integré dans de nombreuses constructions de biosenseurs. Pourtant, elle souffre de nombreux inconvenients. Notamment elle pésente des propriétés photophysiques complexes et une forte sensibilité environnementale qui sont des freins à une interprétation quantitative de ses signaux de fluorescence en imagerie cellulaire. Notre objectif vise à mettre au point, grâce à l'introduction d'un minimum de mutations, un dérivé de la ECFP présentant des propriétés d'émission simplifiées et performantes ainsi qu'une faible sensibilité environementale. Des mutations ont été introduites, par mutagenèse dirigée, à deux positions clefs de la séquence peptidique de la ECFP ce qui a permis de générer des dérivés présentant des propriétés photophysiques et une sensibilité au pH modulées. En particulier, nous avons réussit à générer une protéine fluorescente, l'Aquamarine, aux propriétés photophysiques quasi-idéales caractérisée par un rendement quantique de l'ordre de 0,9 et des déclins d'émission de fluorescence quasi-monexponentiels. Elle présente également une sensibilité au pH fortement réduite avec un pH de demi-transition acide de 3,3.Ce manuscrit présente l'étude détaillée des propriétés d'émission de fluorescence des diverses protéines générées. Plusieurs paramètres présentant un intérêt particulièrement important pour une utilisation adéquate en imagerie de fluorescence ont été évalués. Outre la sensibilité au pH établie sur une large gamme de pH (2,5-11), une attention particulière a été portée sur les performances photophysiques (monoexponentialité du déclin d'émission de fluorescence, durée de vie moyenne, rendement quantique, brillance,...) de ces dérivés. De plus, grâce à des expériences de dichroïsme circulaire, des informations sur les changements structuraux, dont ces dérivés sont le siège à pH très acide, ont été obtenues. Enfin, l'examen détaillé des données spectroscopiques stationnaires et résolues en temps a permis de mettre en lumière l'existence de plusieurs espèces émissives contribuant à la photophysique de ces protéines et à l'origine de leur transition acide. L'ensemble de ces résultats constitue une première approche pour une meilleure compréhension de la relation structure-photophysique-dynamique de la ECFP et de ces dérivés.