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A novel dual-luciferase monitoring apparatus a thesis /Andrews, Thomas. January 2008 (has links)
Thesis (M.S.) --University of Texas Graduate School of Biomedical Sciences at San Antonio, 2008. / Vita. Includes bibliographical references.
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Construção e caracterização de vírus recombinante de febre amarela expressando o gene repórter da Gaussialuciferase / Construction and characterization of a recombinant vírus of Yellow Fever expressing the repórter gene of Gaussia LuciferaseKassar, Telissa da Cunha January 2013 (has links)
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Previous issue date: 2013 / Fundação Oswaldo Cruz. Centro de Pesquisas Aggeu Magalhães. Recife, PE, Brasil / O vírus da febre amarela (YFV, Yellow Fever Virus), um arbovírus da família Flaviridae,é o agente causador da febre amarela (FA), uma doença aguda, febril, não contagiosa, hemorrágica e potencialmente fatal. O YFV é endêmico em regiões tropicais da América do Sul e África. Apesar de sua significância como um problema de saúde pública, muitos mecanismos moleculares da biologia do YFV, como replicação do genoma e patogênese viral ainda não foram bem compreendidos. Avanços em genética reversa viral tem permitido a elucidação de mecanismos da biologia e comportamento viral, bem como a construção de vetores vacinais e desenvolvimento de drogas antivirais. No presente trabalho, descrevemos a construção e caracterização de um vírus recombinante de FA expressando o gene repórter da Gaussialuciferase (GLuc). Utilizando o sistema de recombinação homóloga em levedura, o gene repórter da Proteína Fluorescente Amarela (YFP, Yellow Fluorescent Protein) do vírus recombinante YFV-YFP-DENV1linker, previamente construído em nosso laboratório, foi substituído pelo gene repórter GLuc. A construção foi confirmada por PCR. Os RNAs virais genômicos foram sintetizados in vitro, e posteriormente transfectados em células BHK-21.As células transfectadas foram avaliadas por imunofluorescência indireta e mensuração do gene repórter GLuc. Dois clones foram recuperados e caracterizados em cultivo celular. Nós acreditamos que este vírus repórter deverá ser útilna triagem e desenvolvimento de drogas antivirais específicas, estudos de replicação virale competência vetorial, além da possível utilização como vetor viral vacinal
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Aspectos evolutivos da bioluminescência de elateroidea (coleoptera) do BrasilArnoldi, Frederico Gonzalez Colombo [UNESP] 06 February 2009 (has links) (PDF)
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arnoldi_fgc_dr_rcla.pdf: 2897071 bytes, checksum: fa1429945e5319eef834777ced11123a (MD5) / A partir dos coleópteros luminescentes, mais de 20 luciferases já foram clonadas e seqüenciadas. Dessas, a maioria é de lampirídeos das regiões Neártica e Paleártica, quatro de elaterídeos jamaicanos e uma de um fengodídeo asiático. Apenas outras cinco são oriundas da América do Sul, o continente mais rico em espécies de coleópteros luminescentes e o provável berço evolutivo de Lampyridae, a família com maior número de coleópteros luminescentes. Espécies desta região apresentam a maior variedade de cores de bioluminescência. No presente trabalho clonamos e seqüenciamos luciferases dos gêneros Brasilocerus sp., Phrixothrix sp. e Taximastinocerus sp., da família Phengodidae. Com base na análise filogenética dessas luciferases e outras já publicadas, concluímos que as luciferases das lanternas laterais e cefálicas são codificadas por genes parálogos, e propusemos um modelo para a evolução das cores da bioluminescência nessa família. Também determinamos o genoma mitocondrial de Pyrophorus divergens, membro da família Elateridae. A partir desse genoma e outros já publicados, analisamos a evolução da bioluminescência na superfamília Elateroidea sensu Lawrence e Newton (1995) e concluímos que essa pode ter surgido três vezes independentemente nesse grupo. / From luminescent Coleopteran's, more than 20 luciferases have already been cloned and sequenced. Among them, most part is from lampyrids of Neartic and Paleartic regions, four are from Jamaican elaterids and one is from Asiatic phengodids. Just five are from South American species, the richest continent in luminescent Coleopteran's and, probably, the evolutionary cradle of Lampyridae. Species from this region display the greatest range of bioluminescence colors. At the present work, we cloned and sequenced luciferases from the genera Brasilocerus sp., Phrixothrix sp. and Taximastinocerus sp., from Phengodidae family. Based on the phylogenetic analysis of these genes and other already published, we concluded that head and lateral lantern luciferases are coded by paralogous genes, and we also proposed a model for bioluminescence color evolution in this family. We also sequenced the mitochondrial genome of Pyrophorus divergens, an Elateridae member. Based on this genome and other already published, we analysed the evolutionary history of bioluminescence in Elateroidea superfamily sensu Lawrence e Newton (1995) and concluded that it could have appeared three times independently in this group.
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Characterization of a murine gammaherpesvirus in vitro latency systemMutyambizi, Kudakwashe 04 January 2010 (has links)
The human gammaherpesviruses EBV and KSHV realize their oncogenic potential during latent infection. The species specificity of the human gammaherpesviruses has hindered the study of latency in animal models. Murine gammaherpesvirus MHV-68 (MHV-68) may be used as a representative gammaherpesvirus for the study of latency. The goal was to establish an in vitro model of MHV-68 latency using replication defective MHV-68. ORF50 has been identified as the major viral trans-activator essential for entry into the lytic replication cycle and necessary and sufficient for reactivation of MHV-68 virus from latency. ORF50 null mutants (A50) can theoretically be used to infect cells in vitro to facilitate an analysis of virus gene expression and episome maintenance during latency. In this project A50 mutants containing the luciferase or green fluorescence protein (GFP) under OW50 promoter control were used to infect a variety of cell types. 3T3 fibroblasts are a permissive cell line and were used for an initial characterization of the ability of A50 MHV-68 to establish latency. B lymphocytes and macrophages are the major reservoirs of persistence in vivo thus the ability of A50 mutants to establish latency in NSO B and RAW macrophage cell lines was explored. Latency was readily established and maintained in 3T3 and RAW cells. The low infectability of NSO B- cells restricted the utility of this cell line in studies of latency. Examination of patterns of lytic and latent transcription in 3T3 and RAW cells coordinately infected with A50 MHV-68 revealed reactivation efficiencies of 40-60%. Following long-term passage A50 exhibited stable transcription of two latency related genes M2 and ORF73, with episomal maintenance of the viral genome, in the absence of contaminating lytic infection. The results demonstrate the utility of A50 mutants for studies of gammaherpesvirus latency in vivo.
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Visualizing the function and migration of T cellsDugger, Kari J. January 2007 (has links) (PDF)
Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed Feb. 6, 2008). Includes bibliographical references.
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Development of a novel, quantitative assay for determining the rate of activity of antimalarial drugsKhan, Tasmiyah January 2013 (has links)
Malaria, caused by an intracellular Plasmodium parasite, remains a devastating disease, having claimed approximately 655 000 lives worldwide in 2010. The Medicines for Malaria Venture suggests a "single-dose radical cure" as the ideal malaria treatment since rapid clearance of blood-stage parasites and symptom relief improves patient compliance and limits drug resistance. Thus, novel antimalarials should be rapid-acting and assessing their rate of activity is critical to drug discovery. Traditional evaluation of this rate by morphological assessments is flawed by highly subjective, operator-specific interpretations, mainly due to heterogeneous parasite morphology under routine culture conditions. This study aimed to develop an alternative, quantitative assay. Energy is vital for the growth and maintenance of all living organisms. Commercially available kits allow rapid quantification of the cell's energy currency, ATP. Therefore, quantification of parasite ATP shows potential for diagnosing abnormal parasite metabolism and the kinetics of drug action. In this study, a rapid protocol for detecting ATP in Plasmodium falciparum parasites using a luminescence-based kit was developed and optimised. Furthermore, luciferase-expressing transgenic parasites, in which luciferase activity is detected using a similar kit, were acquired. The utility of both methods for evaluating the rate of drug-induced stress was explored using antimalarials with varying modes of action and, presumably, rates of activity. Results showed that parasite ATP remained unchanged, increased or decreased during drug exposure. Morphological examinations by light microscopy and a Recovery assay, aided interpretation of the drug-induced changes in parasite ATP. These investigations suggested that unchanged parasite ATP levels reflect poor drug action, increased ATP levels indicate a stress response and partially compromised viability, while significantly reduced ATP reflects severely compromised viability. Concerning the Luciferase assay, parasite luciferase activity decreased during drug exposure, even in the presence of proteasome inhibitors. Changes in parasite ATP and luciferase activity occurred at rates which suggested that chloroquine is slow-acting, mefloquine has a moderate rate of activity and artemisinin is rapid-acting. These findings are compatible with the expected rates of activity of these established antimalarials. Hence, measurement of parasite ATP and/or luciferase activity may support assessments of parasite health and the kinetics of antimalarial action during drug discovery
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Pushing The Boundaries of Bioluminescence Using Synthetic Luciferins: A DissertationMofford, David M. 11 September 2015 (has links)
Fireflies are beetles that generate yellow-green light when their luciferase enzyme activates and oxidizes its substrate, D-luciferin. This bioluminescent reaction is widely used as a sensitive reporter both in vitro and in vivo. However, the light-emitting chemistry is limited by the properties of the small molecule D-luciferin. Our lab has developed a panel of synthetic luciferin analogs that improve on the inherent characteristics of D-luciferin. My thesis work focuses on harnessing these novel substrates to further expand the utility and molecular understanding of firefly bioluminescence.
The first part of my thesis focuses on using synthetic luciferins to improve bioluminescence imaging beyond what is possible with D-luciferin. Our substrates emit red-shifted light compared to D-luciferin, bringing the wavelength to a range that is more able to penetrate through tissue, but at a cost of lower signal intensity. I developed mutant luciferases that increase the maximal photon flux with the synthetic luciferins over what is achievable with the wild-type luciferase, and furthermore discriminate between substrates based on their chemical structures. Additionally, I have expanded the bioluminescence toolkit by harnessing the intrinsic properties of the luciferins to non-invasively and specifically assay the activity of a single enzyme (fatty acid amide hydrolase) in live mice. Therefore, my work presents an effective way to generally improve upon bioluminescent reporters, but also to measure the activity of a specific enzyme of interest in the context of a living organism.
The second part of my thesis employs synthetic luciferins to more deeply probe the light-emitting chemistry of bioluminescence. Our synthetic substrates reveal latent luciferase activity from multiple luciferase homologs that are inactive with D-luciferin. These enzymes, the fatty acyl-CoA synthetases, are predicted to be luciferase’s evolutionary predecessors, but it was not clear how the light emitting chemistry originated. My work shows that the luciferase must activate the luciferin and provide oxygen access, but the light emitting chemistry is a fundamental property of that activated intermediate. In summary, the work described herein not only expands our understanding of firefly bioluminescence, but also broadens its practical applications to shine bioluminescent light on the dark corners of biology.
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Desenvolvimento de biossensores raciométricos bioluminescentes de pH e metais divalentes baseados na engenharia da região sensível ao pH nas luciferases de vagalumes / Developing of bioluminescent ratiometric biosensors for pH and divalent metals based on the engineering of the pH-sensing moiety of firefly luciferasesGabriel, Gabriele Verônica de Mello 01 December 2017 (has links)
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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
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Etablierung eines Plasmid-basierten Testsystems zur funktionellen Messung der zellulären Doppelstrangbruch-Reparaturfähigkeit in hämatopoetischen Zellen / functional double-strand-break repair analysis in haematopoietic cellsHermann, Julia 08 March 2011 (has links)
No description available.
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The Three-Dimensional Structure of the Cystic Fibrosis Locus: A DissertationSmith, Emily M. 18 November 2014 (has links)
The three dimensional structure of the human genome is known to play a critical role in gene function and expression. I used chromosome conformation capture (3C) and 3C-carbon copy (5C) techniques to investigate the three-dimensional structure of the cystic fibrosis transmembrane conductance regulator (CFTR) locus. This is an important disease gene that, when mutated, causes cystic fibrosis. 3C experiments identified four distinct looping elements that contact the CFTR gene promoter only in CFTR-expressing cells. Using 5C, I expanded the region of study to a 2.8 Mb region surrounding the CFTR gene. The 5C study shows 7 clear topologically associating domains (TADs) present at the locus, identical in all five cell lines tested, regardless of gene expression status. CFTR and all its known regulatory elements are contained within one TAD, suggesting TADs play a role in constraining promoters to a local search space. The four looping elements identified in the 3C experiment and confirmed in the 5C experiment were then tested for enhancer activity using a luciferase assay, which showed that elements III and IV could act as enhancers. These elements were tested against a library of human transcription factors in a yeast one-hybrid assay to identify potential binding proteins. Element III gave two strong candidates, TCF4 and LEF1. A literature search supported these transcription factors as playing a role in CFTR gene expression. Overall, this work represents a model locus that can be used to test important questions regarding the role of three dimensional looping on gene expression.
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