The goal of this project was to incorporate bacteriophage with Bioluminescent Assay in Real-Time Loop-mediated isothermal amplification (BART-LAMP) for the rapid detection of Mycobacterium avium subspecies paratuberculosis (MAP). As the causative agent of Johne’s Disease, there are no rapid detection methods that are suitable in specificity and sensitivity. A screening assay for phage isolation was developed, and over 400 samples were screened for the isolation of a bacteriophage against MAP. One novel Mycobacterium phage was isolated and characterized using transmission electron miscroscopy, host range studies, restriction enzyme digestion, and pH and temperature stability. It was sequenced, annotated, and underwent an in silico protein analysis. No pathogenic or lysogenic genes were detected, and it was found to be related to Gordonia phage GTE2. BART-LAMP was applied to the detection of the isolated phage using purely extracted DNA and crude phage lysate, showing that phages could be detected successfully. / Beef Cattle Research Council; Agriculture and AgriFood Canada through Growing Forward initiative

This master thesis deals with design and construction of device for optic biosensor. The device are using for detected halogen compound in the water. For excitation testing sample is used LED diode about wave length 590 nm. Photocounter is used for detection of the emitted light after fluorescence Such as fluorescent marker are 5(6)-carboxynaphthofluorescein. The thesis is divided into three head parts. The first part includes the theory needed for realization; the second part takes in detail description of the construction and function of the device and the last part describes measurements with the device.

Despite significant advances in cancer treatment, clinical response remains suboptimal and there is a continued requirement for improved chemotherapeutics. The attrition rate for new therapies is high, due principally to lack of in vivo efficacy and poor pharmacodynamics. Consequently better systems are required to determine in vivo preclinical efficiency and drug-target interactions. Engineering of cancer cells to express fluorescent or bioluminescent proteins, either endogenously or under the control of specific gene promoters, and their detection by noninvasive optical imaging has the potential to improve preclinical drug development. In this study, a panel of colorectal cancer cell lines were engineered to express fluorescent and luminescent proteins either constitutively or under control of gene-promoters for the DNA damage response gene p53 or the cell cycle regulator p21, both important pharmacodynamic sensors. These cell lines were characterised for their potential as in vivo models of primary and metastatic tumour therapy response, several showing significant potential. In addition to the development of these models, this study also addressed the pharmacokinetics of different luciferase substrates and identified optimal temporal and dose characteristics for each. Furthermore, a new application for bioluminescent imaging was developed and validated for use in preclinical evaluation of vascular disrupting agents, a new generation of cancer therapeutic. This study demonstrates that despite the dynamic and variable nature of fluorescent and bioluminescent imaging, reproducible results can be obtained if appropriate precautions are taken. The models developed herein will expedite cancer drug development whilst reducing and refining the use of animals in research.

No description available.

Both insect parasitic/entomopathogenic nematodes and plant parasitic nematodes are of great economic importance. Insect parasitic/entomopathogenic nematodes provide an environmentally safe and effective method to control numerous insect pests worldwide. Alternatively, plant parasitic nematodes cause billions of dollars in crop loss worldwide. Because of these impacts, it is important to understand how these nematodes evolve, and, in the case of entomopathogenic nematodes, how their bacterial symbionts evolve. This dissertation contains six chapters. Chapter one is a review of DNA markers and their use in the phylogenetic systematics of entomopathogenic and insect-parasitic nematodes as well as a review of phylogenetic, co-phylogenetic, and population genetic methodologies. Chapter two characterizes positive destabilizing selection on the luxA gene of bioluminescent bacteria. Our data suggests that bacterial ecology and environmental osmolarity are likely driving the evolution of the luxA gene in bioluminescent bacteria. Chapter 3 examines relationships among bacteria within the genus Photorhabdus. Our analyses produced the most robust phylogenetic hypothesis to date for the genus Photorhabdus. Additionally, we show that glnA is particularly useful in resolving specific and intra-specific relationships poorly resolved in other studies. We conclude that P. asymbiotica is the sister group to P. luminescens and that the new strains HIT and JUN should be given a new group designation within P. asymbiotica. Chapter 4 characterizes the morphology of the head and feeding apparatus of fungal feeding and insect infective female morphs of the nematode Deladenus siricidicola using scanning electron microscopy. Results showed dramatic differences in head, face, and stylet morphology between the two D. siricidicola female morphs that were not detected in previous studies using only light microscopy. Chapter five utilizes comparative transciptomics to identify putative plant and insect parasitism genes in the nematode Deladenus siricidicola. Results from this study provide the first transcriptomic characterization for the nematode Deladenus siricidicola and for an insect parasitic member of the nematode infraorder Tylenchomorpha. Additionally, numerous plant parasitism gene homologues were discovered in both D. siricidicola libraries suggesting that this nematode has co-opted these plant parasitism genes for other functions. Chapter six utilizes a phylogenomic approach to estimate the phylogeny of the nematode infraorder Tylenchomorpha.

This thesis project serves to fill experimental gaps needed to advance the goal of performing pre-clinical trials using an orthotopic rat glioblastoma model to evaluate the efficacy of high-frequency electroporation (H-FIRE) and QUAD-CTX tumor receptor-targeted cytotoxic conjugate therapies, individually and in combination, in selectively and thoroughly treating glioblastoma multiforme. In order to achieve this, an appropriate model must be developed and characterized. I have transduced F98 rat glioma cells to express red-shifted firefly luciferase, which will facilitate longitudinal tumor monitoring in vivo through bioluminescent imaging. I have characterized their response to H-FIRE relative to DI TNC1 rat astrocytes. I have demonstrated the presence of the molecular targets of QUAD in F98 cells. The in vitro characterization of this model has enabled preclinical studies of this promising glioblastoma therapy in an immunocompetent rat model, an important step before advancing ultimately to clinical human trials. / Master of Science / Treating glioblastoma multiforme (GBM), a form of cancer found in the brain, has not been very successful; patients rarely live two years following diagnosis, and there have been no major breakthrough advances in treatment to improve this outlook for decades. We have been working on two treatments which we hope to combine. The first is high-frequency electroporation (H-FIRE), which uses electrical pulses to kill GBM cells while leaving healthy cells alive and blood vessels intact. The second is QUAD-CTX, which combines a toxin with two types of protein that attach to other proteins that are more common on the surface of GBM cells than healthy cells. We have shown these to be effective at disproportionately killing human GBM cells growing in a lab setting. Before H-FIRE and QUAD-CTX may be tested on humans, we need to show them to be effective in an animal model, specifically rats. I have chosen rat glioma cells that will behave similarly to human GBM and a rat species that will not have an immune response to them. I have made these cells bioluminescent so that we may monitor the tumors as they grow and respond to our treatments. I have also shown that QUAD-CTX kills these rat glioma cells, as does H-FIRE. Because of this work, we are ready to begin testing these two treatments in rats.

No description available.

Les bioréacteurs de grandes dimensions, en raison de phénomènes de transfert limitant, sont le siège d’hétérogénéités se traduisant par des gradients locaux de concentration et température. Les microorganismes circulant au sein de ces bioréacteurs subissent donc des fluctuations environnementales qui peuvent affecter leur comportement aux niveaux métaboliques et/ou moléculaires. La réponse microbienne est fonction de la nature, de l’intensité, de la fréquence et de la durée de la perturbation. L’objectif de ce travail est l’étude quantitative de l’impact de l’intensité, la fréquence et l’amplitude d’un stress nutritionnel sur le comportement dynamique d’Escherichia coli, à savoir des ajouts pulsés de glucose lors de cultures continues en régime permanent. Un effort particulier est consacré au développement et à la validation des outils expérimentaux indispensables pour une caractérisation rigoureuse des dynamiques de réponses transitoires sur des échelles de temps allant de secondes à quelques minutes. Pour permettre le suivi in situ et en temps réel des changements métaboliques et moléculaires, une souche bioluminescente est mise en œuvre. Les réponses transitoires sont caractérisées par les vitesses spécifiques, les rendements, les profils d’induction transcriptionnelle, les temps caractéristiques. Selon les différents scenarii réalisés, l’ajustement du métabolisme face aux hétérogénéités de substrat est quantifié selon des échelles de temps aux niveaux macroscopiques et/ou moléculaires ; ces résultats originaux contribuent ainsi à l’implémentation des connaissances sur les interactions dynamiques entre les phénomènes biologiques et les phénomènes physiques ; l’enjeu réside à terme en l’amélioration des processus d’optimisation et d’extrapolation des bioprocédés par l’identification et la quantification des dynamiques des phénomènes limitants / Ineffective mixing entailing heterogeneity issues within industrial bioreactors have been reported to affect microbial metabolisms at cellular and/or molecular levels. Substrate gradients inside large-scale bioreactors are common environmental fluctuations that microorganisms would have to encouter along with the bioprocess. Depending on intensity, frequency and duration of those fluctuations, microorganisms may respond in a different manner. The objective of this work is to study the impact of intensity, frequency and amplitude of glucose perturbations on the dynamics of Escherichia coli responses. An E. coli bioluminescent strain is used for in situ and real-time monitoring of both metabolic and transcriptional changes. For this purpose, short-term glucose excess was simulated, using pulse-based experiments into glucose-limited chemostat cultures. In addition, an important effort is devoted to the development and validation of technical and mathematical tools in order to acquire quantitative and kinetic data on time scales from seconds to minutes. The transient responses are characterized, using specific rates, yields, transcriptional induction profiles and characteristic response times, and are compared in the different defined perturbation scenarios. The results reflected the fact that short-term heterogeneities of substrate affect both cell metabolism and regulation at macroscopic and/or molecular levels. Quantitative understandings of the dynamics during transient responses to environmental perturbations can thus shed light on the bioprocess optimization

Submitted by Gabriele Gabriel (gabriele.mgabriel@yahoo.com.br) on 2018-01-05T14:37:40Z No. of bitstreams: 1 Tese_Gabriele-Gabriel_VERSAO-FINAL.pdf: 3365844 bytes, checksum: c94734430443f3bf6c8ad3ad9fd11a8d (MD5) / Rejected by Ronildo Prado (bco.producao.intelectual@gmail.com), reason: Oi Gabriele, Faltou enviar a Carta comprovante assinada pelo orientador. Solicite o modelo em sua Secretaria de Pós-graduação, preencha e colete a assinatura com o orientador e acesse novamente o sistema para fazer o Upload. Fico no aguardo para finalizarmos o processo. Abraços Ronildo on 2018-01-18T16:17:33Z (GMT) / Submitted by Gabriele Gabriel (gabriele.mgabriel@yahoo.com.br) on 2018-01-19T17:45:54Z No. of bitstreams: 2 GABRIEL_Gabriele_2017.pdf: 4796378 bytes, checksum: b47c6fae3bd085c69ed8a81c74a9be4d (MD5) GABRIEL_Gabriele_carta.pdf: 557241 bytes, checksum: fbed02fe69c0cab6190201e18b304f40 (MD5) / Approved for entry into archive by Ronildo Prado (bco.producao.intelectual@gmail.com) on 2018-01-22T18:44:07Z (GMT) No. of bitstreams: 2 GABRIEL_Gabriele_2017.pdf: 4796378 bytes, checksum: b47c6fae3bd085c69ed8a81c74a9be4d (MD5) GABRIEL_Gabriele_carta.pdf: 557241 bytes, checksum: fbed02fe69c0cab6190201e18b304f40 (MD5) / Approved for entry into archive by Ronildo Prado (bco.producao.intelectual@gmail.com) on 2018-01-22T18:44:18Z (GMT) No. of bitstreams: 2 GABRIEL_Gabriele_2017.pdf: 4796378 bytes, checksum: b47c6fae3bd085c69ed8a81c74a9be4d (MD5) GABRIEL_Gabriele_carta.pdf: 557241 bytes, checksum: fbed02fe69c0cab6190201e18b304f40 (MD5) / Made available in DSpace on 2018-01-22T18:51:21Z (GMT). No. of bitstreams: 2 GABRIEL_Gabriele_2017.pdf: 4796378 bytes, checksum: b47c6fae3bd085c69ed8a81c74a9be4d (MD5) GABRIEL_Gabriele_carta.pdf: 557241 bytes, checksum: fbed02fe69c0cab6190201e18b304f40 (MD5) Previous issue date: 2017-12-01 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Bioluminescence, the emission of visible light by living organisms is widely used in biosensors. Firefly luciferases and genes are among the most used reporter gene in bioluminescent biosensors. Firefly luciferases are pH-sensitive, exhibiting a red shifted bioluminescence spectra on the presence of metals, high temperatures and acidic pH, being this last property considered unusual for the most analytical applications. Nowadays most luminescent biosensors to metals and pH are fluorescent, and few of them are ratiometric based on spectral changes. Bioluminescent biosensors despite been less common, have same advantages as low background and does not need UV light irradiation, bring no photodamage to the cell. The aim of this project was: (I) study the applicability of the use of Macrolampis sp2 firefly luciferase and other pH-sensitive luciferases as spectral intracellular ratiometric pH biosensor; (II) apply these pH ratiometric biosensors in mammalian cells to investigate its physiology in real time and (III) developing, by engineering the pH sensor region of Macrolampis sp2 firefly luciferase, a ratiometric biosensor specific to metals. We obtained a relation between the pH and the ratio of the bioluminescence at green and red region of the spectra, allowing estimate ratiometrically the intracellular pH of bacteria and mammalian cells. Besides that, we confirmed its use to cellular image of pH and observed that occurs an alkalinization of the cytosol and nucleus during cell division and apoptosis. We demonstrate the applicability of firefly luciferases, in special the luciferase of Macrolampis sp2, as a ratiometric biosensor to metals and intracellular pH. The existence of a linear relationship between the concentrations of metals like cadmium, mercury and zinc, and the ratio of the bioluminescence at green and red region of the spectra, allowed also estimate ratiometrically, for the first time, concentrations of metals less than 100 µM, enabling the use of firefly luciferases as bioavailability indicator to toxic and potential toxic metals. / Bioluminescência, a emissão de luz fria e visível por organismos vivos é amplamente utilizada em biossensores. Luciferases de vagalumes e seus genes estão entre os genes repórteres mais utilizados em biossensores bioluminescentes. Luciferases de vagalumes são sensíveis ao pH, apresentando um deslocamento do espectro de bioluminescência para o vermelho na presença de metais, em temperaturas elevadas e pH ácido, sendo esta última propriedade considerada sem utilidade para a maioria das aplicações analíticas. Atualmente a maioria dos biossensores luminescentes para metais e pH são fluorescentes, poucos deles são raciométricos baseados nas mudanças espectrais. Biossensores bioluminescentes apesar de serem menos comuns, possuem algumas vantagens como baixo background e não necessitam de irradiação com luz UV, não causando fotodanos às células. Os objetivos desse projeto foram: (I) investigar a aplicabilidade de uso da luciferase do vagalume Macrolampis sp2 e outras luciferases pH-sensitivas como biossensor espectral raciométrico intracelular de pH; (II) aplicar esses biossensores raciométricos de pH em células de mamíferos para investigar sua fisiologia em tempo real e (III) desenvolver, por engenharia da região sensora de pH da luciferase de Macrolampis sp2, um biossensor raciométrico específico para metais. Obtivemos uma relação entre o pH e a razão da bioluminescência nas regiões do verde e do vermelho do espectro, permitindo estimar raciometricamente o pH intracelular de bactérias e células de mamíferos. Além disso, confirmamos seu uso para imagem celular de pH, e observamos que ocorre uma alcalinização do citosol e núcleo durante os processos de divisão celular e apoptose. Demonstramos a aplicabilidade das luciferases de vagalumes, em especial a luciferase de Macrolampis sp2, como biossensor raciométrico para metais e pH intracelular. A existência de uma relação linear entre a concentração de metais como cádmio, mercúrio e zinco, e a razão da bioluminescência nas regiões do verde e do vermelho do espectro, permitiu também estimar raciometricamente pela primeira vez concentrações de metais menores que 100 µM, possibilitando o uso de luciferase de vagalumes como indicador de biodisponibilidade de metais tóxicos e potencialmente tóxicos. / FAPESP: 2014/04477-9 / FAPESP: 2015/22603-4 / FAPESP: 2016/15946-5

No description available.