No description available.

Yersinia ruckeri is the bacterial agent causing enteric redmouth disease (ERM) in rainbow trout leading to economic losses in intensive aquaculture. There are two main serovars and several minor groups based on O-antigens. The first goal of this thesis was to examine the difference between serotypes of Y. ruckeri in the course of infection in fish by applying in vivo bioluminescent imaging in rainbow trout (Oncorhynchus mykiss) and coho salmon (Oncorhynchus kisutch). In infection trials, the bioluminescent strains were infective, but the bioluminescent signal was not detected in fish infected with bacterial loads of 107 colony forming units per gram of kidney tissue. Skin and scales and the kidney blocked the luminescent signal emitted from the bacteria. The second goal of this thesis was to identify genetic markers which correlate with traditional O-antigen serotyping reactions. Using the sequences of genes which are part of the lipopolysaccharide biosynthetic pathway, oligonucleotide primers were designed to be complimentary to a fragment of wzx, the O-antigen flippase, and to wzy, the O-antigen polymerase, of serotype 1 Y. ruckeri strain RS 11. When these primers were used in polymerase chain reaction, an 1183 bp fragment of wzx and a 755 bp fragment of wzy were seen with DNA from 8 serovar 1 and 9 serovar 1a strains and not from other serovars identified by rabbit anti-sera agglutination. Southern blotting suggested there was little homology between serovar 1 wzx and wzy, and the same genes of the remaining serovars if present.

As the move towards the miniaturization of many diagnostic and detection systems continues, the need for increasingly versatile yet sensitive labels for use in these systems also grows. Luminescent reporters provide us with a solution to many of the issues at hand through their unique and favorable characteristics. Bioluminescent proteins offer detection at extremely low concentrations and no interference from physiological fluids leading to excellent detection limits, while the vast number of fluorescent proteins and molecules available allows the opportunity to select a tailored reporter for a specific task. Both provide relatively simply instrumentation requirements and have exhibited great promise with many of the miniaturized systems such as lab-on-a-chip and lab-on-a-CD designs. Herein, we describe the novel employment of luminescent reporters for four distinct purposes. First off, by combining both time and wavelength resolution we have expanded the multiplexing capabilities of the photoprotein aequorin beyond duel-analytes, demonstrating the ability to simultaneously detect three separate analytes. Three semi-synthetic aequorin proteins were genetically conjugated to three pro-inflammatory cytokines (interleukins 1, 6, and 8) resulting in aequorin labeled cytokines with differing emission maxima and half lives to allow for the simultaneous detection of all three in a single solution through the elevated physiological concentration range. Secondly a semi-synthetic aequorin variant has been genetically enhanced to serve as an immunolabel and exhibited the ability to sensitively detect the acute myeloid leukemia marker, CD33, down to the attomole level in addition to improving aequorin imaging capabilities. In the third example, the aequorin complex was rationally, genetically split into two parts and attached to the termini of the cAMP selective cAMP receptor protein (CRP) creating a genetically fused molecular switch. The conformational change experienced by CRP upon the binding of cAMP translates into a loss of bioluminescent signal from aequorin and has shown the ability to respond linearly to cAMP over several orders of magnitude. Lastly, through custom design, a reagentless, portable, fluorescent fiber optic detection system has been developed, capable of being integrated into the body through a heart catheter. The system was able to respond to changes in potassium concentration selectively, reproducibly and reversibly with a fast response time of one minute.

Multiple myeloma (MM) is an incurable plasma cell malignancy. NK cells have demonstrated anti-MM activity in allogeneic transplants and donor lymphocyte infusions, and may provide a more effective therapy for MM. This work demonstrates cytotoxicity of NK-92 and KHYG-1 against MM cells in chromium release and flow cytometry cytotoxicity assays. At a 10:1 effector to target ratio, the cytotoxicity of NK cell lines against MM cells is 50-90%. Blocking NKp30 significantly reduces the cytotoxicity of NK-92 and KHYG-1, while blocking NKG2D and DNAM-1 only reduces the cytotoxicity of NK-92. Notably, NK-92 and KHYG-1 have shown preferential cytotoxicity against the clonogenic population, killing 89-99% in a methylcellulose cytotoxicity assay. Preliminary results in a xenograft bioluminescent mouse model show that NK-92, but not KHYG-1, reduces the tumor burden detected by bioluminescence imaging and bone marrow engraftment by flow cytometry. Therefore, NK cell lines may offer a more effective therapy for MM.

Multiple myeloma (MM) is an incurable plasma cell malignancy. NK cells have demonstrated anti-MM activity in allogeneic transplants and donor lymphocyte infusions, and may provide a more effective therapy for MM. This work demonstrates cytotoxicity of NK-92 and KHYG-1 against MM cells in chromium release and flow cytometry cytotoxicity assays. At a 10:1 effector to target ratio, the cytotoxicity of NK cell lines against MM cells is 50-90%. Blocking NKp30 significantly reduces the cytotoxicity of NK-92 and KHYG-1, while blocking NKG2D and DNAM-1 only reduces the cytotoxicity of NK-92. Notably, NK-92 and KHYG-1 have shown preferential cytotoxicity against the clonogenic population, killing 89-99% in a methylcellulose cytotoxicity assay. Preliminary results in a xenograft bioluminescent mouse model show that NK-92, but not KHYG-1, reduces the tumor burden detected by bioluminescence imaging and bone marrow engraftment by flow cytometry. Therefore, NK cell lines may offer a more effective therapy for MM.

No description available.

No description available.

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.

Esta tese descreve estudos realizados na tentativa de purificar e caracterizar enzimas envolvidas na BL de fungos, além de trabalhos conduzidos a fim de investigar o mecanismo da bioluminescência de fungos. Inicialmente, tentou-se isolar as duas enzimas supostamente responsáveis pala reação bioluminescente em fungos. Parâmetros de atividade ótima (pH e temperatura) e comportamento cinético foram investigados. Todavia, com a descoberta de que a luciferina fúngica é o derivado hidroxilado da hispidina (3-hidróxihispidina), novas estratégias foram abordadas. Os esforços se concentraram na purificação da luciferase, visto que a hidroxilase não faz parte do sistema bioluminescente de fungos. Avaliação da interação da luciferase fúngica com a luciferina ou derivados dela sugeriram comportamento relativamente promíscuo da enzima. Os resultados indicaram que a reação luciferina-luciferase é favorecida em meio básico (pH ~8), a ~20 °C. Ensaios com 18O2 revelaram que a inserção de oxigênio na molécula de luciferina produz um intermediário cuja descarboxilação gera a oxiluciferina. Paralelamente, a síntese da hispidina in vitro a partir de ácido cafeico na presença de malonil-CoA e de extrato de micélios bioluminescentes resultou na emissão de luz, confirmando que a luciferina é reciclada no processo. / This work describes studies performed to purify and characterize enzymes responsible for the fungal bioluminescence. Also, it shows important data that contributes to understand the mechanism for bioluminescence reaction in fungi. First, we tried to isolate two enzymes suspected of being involved on fungal bioluminescence. Optimum activity parameters (pH and temperature) and kinetic behavior were investigated. However, the discovery that fungal luciferin is the hispidin derivative 3-hydroxyhispidin demanded adaptations in the project. First of all, concentrates efforts to luciferase purification was priority, since hydroxylase is not part of the bioluminescent system of fungi. Studies on the luciferase interaction with different substrates showed some promiscuity for the enzyme. The results indicated higher intensity of light from luciferin-luciferase reaction in alkaline solutions (pH ~ 8) at ~ 20 °C. The reaction in medium with 18O2 revealed that insertion of oxygen into the luciferin structure produces an intermediate whose decarboxylation generates oxyluciferin. In parallel, the in vitro synthesis of hispidin using caffeic acid and malonyl-CoA with the mycelium extract resulted in the emission of light, confirming that luciferin is recycled in the process.

Esta tese descreve estudos realizados na tentativa de purificar e caracterizar enzimas envolvidas na BL de fungos, além de trabalhos conduzidos a fim de investigar o mecanismo da bioluminescência de fungos. Inicialmente, tentou-se isolar as duas enzimas supostamente responsáveis pala reação bioluminescente em fungos. Parâmetros de atividade ótima (pH e temperatura) e comportamento cinético foram investigados. Todavia, com a descoberta de que a luciferina fúngica é o derivado hidroxilado da hispidina (3-hidróxihispidina), novas estratégias foram abordadas. Os esforços se concentraram na purificação da luciferase, visto que a hidroxilase não faz parte do sistema bioluminescente de fungos. Avaliação da interação da luciferase fúngica com a luciferina ou derivados dela sugeriram comportamento relativamente promíscuo da enzima. Os resultados indicaram que a reação luciferina-luciferase é favorecida em meio básico (pH ~8), a ~20 °C. Ensaios com 18O2 revelaram que a inserção de oxigênio na molécula de luciferina produz um intermediário cuja descarboxilação gera a oxiluciferina. Paralelamente, a síntese da hispidina in vitro a partir de ácido cafeico na presença de malonil-CoA e de extrato de micélios bioluminescentes resultou na emissão de luz, confirmando que a luciferina é reciclada no processo. / This work describes studies performed to purify and characterize enzymes responsible for the fungal bioluminescence. Also, it shows important data that contributes to understand the mechanism for bioluminescence reaction in fungi. First, we tried to isolate two enzymes suspected of being involved on fungal bioluminescence. Optimum activity parameters (pH and temperature) and kinetic behavior were investigated. However, the discovery that fungal luciferin is the hispidin derivative 3-hydroxyhispidin demanded adaptations in the project. First of all, concentrates efforts to luciferase purification was priority, since hydroxylase is not part of the bioluminescent system of fungi. Studies on the luciferase interaction with different substrates showed some promiscuity for the enzyme. The results indicated higher intensity of light from luciferin-luciferase reaction in alkaline solutions (pH ~ 8) at ~ 20 °C. The reaction in medium with 18O2 revealed that insertion of oxygen into the luciferin structure produces an intermediate whose decarboxylation generates oxyluciferin. In parallel, the in vitro synthesis of hispidin using caffeic acid and malonyl-CoA with the mycelium extract resulted in the emission of light, confirming that luciferin is recycled in the process.