Indiana University-Purdue University Indianapolis (IUPUI) / MicroRNAs (miRNA) are short, 18-24 nucleotide long noncoding RNAs. These small RNAs, which are initially transcribed in the nucleus, are transported into the cell cytoplasm where they regulate protein translation either through direct cleavage of mRNA, or indirect inhibition through binding to mRNA and disrupting the protein translation machinery. Recently, miRNAs have gained much attention due to their implication in numerous diseases and cancers. It has been found that heightened or lowered levels of miRNA in diseased cells vs. healthy cells are linked to disease progression. It is therefore immensely important to be able to detect these small molecules. Current detection methods of Northern blotting, microarrays, and qRT-PCR suffer from drawbacks including low sensitivity, a lack of simplicity, being semi-quantitative in nature, time-consuming, and requiring expensive instruments. This work aims to develop novel miRNA technologies which will address these above problems. Bioluminescent labels are promising alternatives to current methods of miRNA detection. Bioluminescent labels are relatively small, similar in size to fluorescent proteins, and they emit very intense signals upon binding to their substrate. Bioluminescent labels are advantageous to fluorescent labels in that they do not require an external excitation source, rather, the excitation energy is supplied through a biochemical reaction. Therefore, background signal due to excitation is eliminated. They also have the advantage of being produced in large amounts through bacterial expression. Four miRNA detection methods are presented which utilize luminescence-based methods. Three employ Renilla luciferase, a bioluminescent protein, and one is based on fluorescence. The presented methods are capable of detecting miRNA from the picomole (nanomolar) level down to the femtomole (picomolar) level. These methods are rapid, sensitive, simple, and quantitative, can be employed in complex matrices, and do not require expensive instruments. All methods are hybridization-based and do not require amplification steps.

The preservative challenge test is a regulatory requirement specified in various pharmacopoeias to determine the efficacy of preservatives. However, such testing is a labour-intensive repetitive task and often requires days before results can be generated. Microbial biosensors have the potential to provide a rapid and automated alternative to the traditional viable counting currently in use. However, the selection of appropriate promoters is essential. The bioluminescent reporter strains used in the current study comprise the Photorhabdus luminescence lux CDABE reporter genes under the control of five individual constitutive Escherichia coli promoters: outer lipoprotein (lpp); twin arginine translocase (tatA); lysine decarboxylase (ldc); lysyl t-RNA (lysS); and ribosomal protein (spc). The promoter plus lux CDABE constructs were cloned, ligated into the plasmid vector pBR322 and transformed into E. coli ATCC 8739. The bioluminescence intensity in the decreasing order of constitutive promoter was lpp > spc> tatA> ldc > lysS. The five biosensor strains tested successfully in PET assays and demonstrated accuracy with a minimum detection limit of 103 CFU/ml, a detection range of 6 orders magnitude, and yielded equivalent results to methods currently recommended by the pharmacopoeias. The bioluminescent biosensors were used to monitor the efficacy of preservatives; sorbic acid at concentrations of 0.031% to 0.2% at pH 5.0, and benzalkonium chloride at concentrations of 0.0062% to 0.00039% alone and in combination with 0.03% EDTA. The 99.9% percentage of bioluminescence reduction of tatA-lux, ldc-lux, lysS-lux, and spc-lux was statistically equivalent to the 3 log10 CFU/ml reduction as required by the Pharmacopeias’. Strong significant correlations between bioluminescence and the methods recommended by the pharmacopoeias were obtained when the biosensor strains were challenged with preservatives, for all except lpp-lux E. coli. The bioluminescence expressed by the lpp-lux biosensor was significantly lower during long-term stationary phase than it was for any of the other biosensors and was also significantly lower than for any of the other biosensors in the presence of preservatives. Since the plasmid copy number and viable counts for lpp-lux did not change under these conditions, it suggests that perhaps lpp-lux was down regulated under stress conditions. There were no statistically significant differences between the results of the bioluminescence assays and the results of the viable count and ATP chemiluminescence assay. Virtual foot printing (using Regulon DB database) demonstrated that two crp binding sites overlapping the -10 regions are located on the negative strand of the lysS promoter sequences and that one crp binding site is located in lpp. The biosensor strains ldc-lux exhibited levels of bioluminescence per cell significantly lower than spc in the presence of preservatives whilst there was a significant increase in bioluminescence per cell by tatA-lux under alkaline conditions (pH 8.9) during long-term stationary phase. Amongst the five biosensor strains tested in the current work, it was determined that the spc-lux strain would be the most attractive candidate for further work, since the bioluminescence expressed per cell was significantly greater, by 10-1000 times, than that expressed by the other four promoters when challenged with the preservatives tested with excellent significant correlations between bioluminescence expression and viable counts in the PET assays with the various preservatives in this study (R2: 8.79-1.00). The bioluminescent biosensor strains showed no statistical differences from the control strains (wildtype E.coli ATCC 8739 and E.coli carrying a promoterless [pBR322.lux] for adneylate energy charge (AEC), plasmid copy number (PCN) bioluminescence or viable counts over 28 days. The emission of bioluminescence by the four bioreporter strains across 28 days is reflected by the stability of PCN with correlations of 0.78-0.90, except for lpp-lux with R2: 0.59. The following promoter elements were found likely to assist greater expression of bioluminescence: an A+T level of approximately 50% between the -40 and -60 regions (the UP element); a G+C level of approximately 50% within the -10 and +1 regions; the extended -10 region and -10 region of consensus sequence RpoD (σ70/D).

Membranes with a pore size rating of 0.2μm are recommended for the sterilisation of liquids by filtration and are validated for this purpose by a retention test with Pseudomonas diminuta. Practices for retention testing were found to vary among the membrane manufacturers and only one type of commercial 0.2μm rated membrane was found to reliably retain P. diminuta. The retention for P. diminuta given by experimental grafted membranes was studied and was sometimes higher than that given by non-grafted membranes due to obstruction of the pores by graft material. The dimensions for individual cells of P. diminuta was studied by scanning electron microscopy and a rapid electronic method. Bacteria of larger dimensions than the pore size rating of experimental membranes were found in test permeates. It was shown that cells from an aerated P. diminuta culture were larger than cells from a similar but stationary culture. A retention test procedure for 0.2 μm rated membranes using cross-flow filtration was developed. The procedure simulated process conditions and enabled tubular ceramic monolithic membranes and flat-sheet membranes to be retention tested with P. diminuta. It is feasible that a standard retention test using cross-flow filtration can be developed. The time needed for results from current retention test procedures to become available is a consequence of using traditional cultural techniques for permeate analysis. Test procedure were developed using three popular methods for the rapid detection and enumeration of bacteria (ATP luminescence, impedance microbiology and the DEFT) for the detection and enumeration of P. diminuta in retention test permeates. The method using ATP luminescence was found to be the most applicable. The development of a bioluminescent strain of P. diminuta through genetic engineering will enable the rapid, sensitive and straightforward retention testing of 0.2 μm rated membranes. Retention tests using a bioluminescent strain of Escherichia coli containing the structural genes for bacterial luciferase indicated that the proposed test is feasible. Developments were made towards cloning the same genes into P. diminuta. The use of all bioluminescent micro-organisms for membrane retention testing is the subject of a patent application and a proposal for a three year SERC research grant.

Après trois pandémies majeures responsables de millions de morts, la peste n'a pas encore disparu. Cette maladie est causée par la bactérie Yersinia pestis, dont les mécanismes de virulence sont encore mal compris. Le suivi d'infection de la peste bubonique chez la souris, méthode classique pour étudier le processus infectieux, requiert beaucoup d'animaux et de temps pour obtenir des résultats significatifs. L'imagerie in vivo et en temps réel par bioluminescence permet de suivre la progression du pathogène au cours du processus infectieux en observant les animaux de façon non invasive. Nous avons transformé la souche virulente CO92 avec le plasmide pEm7-luxCDABE et confirmé la production de bioluminescence in vitro et in vivo. Nous avons pu quantifier la charge bactérienne dans plusieurs organes colonisés sans sacrifier l'animal et établir le schéma de progression de la bactérie au cours de la maladie. Après formation d'un foyer infectieux au site d'injection, la colonisation du ganglion lymphatique inguinal drainant ce site a été observée. Nous avons démontré que la bactérie suit un trajet direct du ganglion lymphatique inguinal au ganglion lymphatique axillaire. L'étape suivante est la colonisation des organes filtrant le sang, puis survient la septicémie dans les phases terminales de la mort. Nous avons établi que la forte variabilité dans le processus infectieux était due au temps pendant lequel la bactérie était contenue au site d'injection. À partir du moment où les ganglions lymphatiques sont colonisés, la cinétique de progression est à la fois régulière et rapide ; la septicémie survient dans les deux jours, suivie de près par la mort.

An emerging trend towards non-laboratory based biological and microbiological marker analysis is occurring in multiple sectors of science and industry. In the medical sector, these trends have demonstrated that conducting sample analyses away from centralised laboratories not only makes analyses quicker and more convenient (e.g. a home pregnancy test), but can offer services that are otherwise impractical (e.g. mobile laboratories to diagnose disease in the developing world). In the environmental sector, similar benefits, plus the ability to develop and test hypotheses, protocols and sampling strategies within a field campaign, are possible with in-field analyses. Icy environments in particular would benefit from in situ or in-field life detection as they are typically remote, and hence impart high logistical costs for repeated field campaigns and associated sample return with the implication that the efficiency of scientific return is poor. Unfortunately, most equipment and protocols developed for microbiological analyses in other sectors of science and industry are unsuitable for direct application to in-field use in icy environments because of poor compatibility with icy environment sample matrices and frequently inappropriate microbiological targets. Hence within this work, two hypotheses were tested: that (i) microbiological detection infield in icy environments is possible and through this (ii) unique and more efficient scientific studies can be conducted. Cont/d.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Master of Science / Department of Anatomy & Physiology / Deryl L. Troyer / Breast cancer ranks second as a cause of cancer death in women in the USA. Detection of early tumors and tumor-targeted treatments could decrease the problems associated with breast cancer management. Photodynamic therapy (PDT) is a cancer treatment that uses a photosensitizer and a specific wavelength of light and is currently in clinical trials for breast cancer. When tumor cells which have absorbed photosensitizer are exposed to the correct wavelength of light, reactive oxygen species are generated, resulting in tumor cell death. Poor tissue penetration of light is a major limitation in PDT, restricting its use to treatment of localized tumors. Light generation at the tumor area might increase the effectiveness of PDT. Polymorphonuclear neutrophils (PMNs) are known to often infiltrate breast adenocarcinoma, and their activatation in tumor stroma produces luminescence in the presence of luminol. Here, we hypothesized that luminol can be used as a theranostic agent for luminescence-based early tumor detection (diagnosis) and in situ PDT (treatment). BALB/c mice were transplanted with 4T1 mammary adenocarcinoma cells to establish a breast adenocarcinoma model. The early tumor detection objective was tested by daily intraperitoneal injection of luminol and in vivo luminescence imaging. To test the PDT treatment objective,the photosensitizer 5-aminolevulinic acid (ALA) and luminol were administered to mice through intraperitoneal and intravenous routes, respectively. This treatment regimen was repeated six times and ALA alone/luminol alone/saline treated tumor-bearing mice were used as controls. Results demonstrated that luminol allowed detection of activated PMNs only two days after 4T1 cell transplantation, even though tumors were not yet palpable. Relative differences in the increase of tumor volume and final tumor weights were analyzed to test the in situ PDT. Analysis of the data showed luminol treatments resulted in breast adenocarcinoma tumor growth attenuation. In conclusion this study provides evidence that luminol can be a theranostic agent for breast adenocarcinoma.

Doctor of Philosophy / Department of Chemistry / Stefan H. Bossmann / The photochromic spirodihydroindolizine/betaine (DHI/B) system has been reinvestigated applying picosecond, microsecond, stationary absorption measurements, and NMR-kinetics. The first surprise was that the electronic structure of the betaines is quite different than commonly assumed. The photochemical ring-opening of DHIs to betaines is a conrotatory 1,5 electrocyclic reaction, as picosecond absorption spectroscopy confirms. The (disrotatory) thermal ring-closing occurs from the cisoid betaine. The lifetime of the transoid betaine is 60 s at 300 K, whereas the lifetime of the cisoid isomer is of the order of 250 microseconds. According to these results, the electrocyclic back reaction of the betaines to the DHI is NOT rate determining, as previously thought, but the cisoid-transoid-isomerization of the betaine. Although the presence of a second nitrogen atom increases the photostability of the spirodihydroindolizine-pyridazine/betaine-system remarkably, the photochemical reaction mechanism appears to be exactly the same for spirodihydroindolizine-pyridazine/betaine-system. A nondestructive photoswitch or an information recording systems has been explored using styryl-quinolyldihydroindolizines. Both isomers DHI and betaine are fluorescent. When the blue betaine is stabilized in a thin polymethyl methacrylate (PMMA) matrix, it is stable for several hours even in room temperature and very stable at 77K. Although irradiation of visible light = 532 nm allows the photo-induced reaction of the Betaine back to the DHI, a nondestructive read-out can be performed at λ = 645 nm upon excitation with λ = 580 nm. Image recording (write) and read-out, as well as information storage (at 77K) have been demonstrated. Charged and maleimide-functionalized DHI/B systems have beed synthesized for use as photochemical gates of the mycobacterial channel porin MspA. Positively charged and maleimide functionalized DHI groups that were attached to the DHI/B-system permit the binding of the photoswitch to selective positions in the channel proteins due to the presence of a cysteine moiety. An inexpensive new method for the large scale synthesis of coelenterazine is developed. A modified Negishi coupling reaction is used to make pyrazine intermediates from aminopyrazine as an economical starting material. This method permits the use of up to 1g coelenterazine per kg body weight and day, which turns the renilla transfected stem cells into powerful light sources.

A bioluminescência desperta o interesse humano há muitos séculos. Presente em quatro dos sete reinos taxonômicos, Monera, Chromista, Animalia e Fungi, cada um com mecanismos muito diferentes. Pode-se dizer que o estudo químico da bioluminescência começou com os experimentos de Dubois no século XIX, que cunhou os termos luciferina e luciferase, termos genéricos para o substrato e enzima envolvidos na reação, respectivamente. No caso específico de fungos, o envolvimento de enzimas foi debatido por quase cinco décadas, após a proposta enzimática de Airth e Foerster na década de 1960 e a não enzimática por Shimomura, em 1989. Somente em 2009 a hipótese de Airth e Foerster foi confirmada pelo nosso grupo, seguido da identificação da luciferina fúngica e o envolvimento de hispidina, como molécula precursora, em 2015 pelo grupo de Yampolsky. Para conseguir elucidar mecanismos químicos, a técnica de espectrometria de massas pode ser empregada para a identificação estrutural de reagentes e intermediários destas e de outras reações orgânicas. Após a confirmação do envolvimento de hispidina na bioluminescência de fungos, utilizou-se a técnica de cromatografia líquida acoplada a espectrometria de massas para identificar a presença de hispidina, seus derivados e intermediários precursores, em fungos e em algumas plantas. / Bioluminescence arises human interest for centuries. Occurring in four of seven taxonomical Kingdoms, Monera, Chromista, Animalia and Fungi, each of them with completely different mechanisms. The chemical study of bioluminescence starts in XIX century with Dubois, who coined the terms luciferin and luciferase, generic terms for substrate and enzyme involved in the bioluminescent reaction, respectively. In the specific case of fungi, enzyme involvement has been debated for almost five decades, after the enzymatic proposal by Airth and Foerster, during the 1960 decade, and the non-enzymatic proposal by Shimomura in 1989. It was only in 2009 when the proposal by Airth and Foerster was confirmed by our group, followed by the identification of the fungal luciferin and the involvement of hispidin, as the precursor molecule, in 2015 by Yampolskys group. To elucidate the chemical mechanisms, mass spectrometry can be employed to structural identification of reagents and intermediates on these and other organic reactions. After the confirmation of hispidin involvement in fungi bioluminescence, liquid chromatography coupled with mass spectrometry was uses do identify the presence of hispidin, its derivatives and precursor intermediates in fungi and selected plants.

Este estudo foi realizado com o objetivo de desenvolvimento e validação do método microbiológico rápido empregando a técnica de bioluminescência de Adenosina Trifosfato (ATP) como método alternativo para o teste de esterilidade. O ATP reage com o sistema enzimático luciferina/luciferase e gera um fóton de luz em presença de íons magnésio, reação que pode ser utilizada na detecção de microrganismos. A luz gerada na reação é medida por um dispositivo chamado luminômetro, que traduz o sinal em unidades relativas de luz (URL), metodologia altamente sensível que pode ser utilizada na análise de produtos estéreis com o objetivo de diminuição no tempo do ensaio. Enquanto a turbidez do meio de cultura só pode ser visualizada quando o contaminante chega à concentração de 106 UFC/ml, a tecnologia de bioluminescência de ATP pode detectar amostras com concentração em torno de 104 UFC. Foram empregados na validação dados obtidos a partir do método tradicional de esterilidade (técnica de filtração) realizado paralelamente ao método alternativo. As soluções parenterais utilizadas nos ensaios foram: solução fisiológica 0,9%; solução de dextrose 5%; ringer lactato; e solução de metronidazol 0,5%. As soluções-teste foram inoculadas intencionalmente com suspensões microbianas preparadas através da diluição de Bioballs&#174, com concentrações de 10 UFC/100 ml, 2 UFC/100 ml e 0,4 UFC/100 ml. Após a realização do teste convencional, as membranas resultantes do ensaio foram incubadas nos meios de cultura caldo caseína de soja e tioglicolato. Alíquotas destes meios foram retiradas após 96 horas de incubação para análise pelo método alternativo. Os seguintes microrganismos foram selecionados para o estudo: Staphylococcus aureus, Bacilus subtilis, Pseudomonas aeruginosa, Candida albicans, Clostridium sporogenes, Aspergillus brasiliensis, Kocuria rosea e Micrococcus luteus. A análise dos resultados obtidos mostrou que o método alternativo é capaz de detectar os microrganismos testados. Quanto à sensibilidade, o método alternativo apresentou vantagem na concentração 2 UFC/100 ml, e equivalência nas outras concentrações. A não interferência dos diferentes produtos e meios nos resultados encontrados permite vislumbrar evidência de robustez do método. Adicionalmente, em relação ao tempo de resposta, o método alternativo demonstrou ser equivalente ao convencional (p-valor=0,43). / This study is being conducted with a goal of validating and developing the fast microbiological method of ATP bioluminescence, as an alternative method to the sterility test. The ATP reacts with the enzymatic system luciferin-luciferase and produces light in the presence of magnesium ions, this reaction can be used for microorganism\'s detection. The light generated in this reaction can be measured by a device called luminometer that translates the signal in relative light units (RLU). This methodology has high sensibility and it can be used in the sterile products analysis with the objective of reducing the time of the sample. While the turbidity of the culture medium it can be visualized just when the sample reaches a concentration of 106 UCF per mL, the bioluminescence assay can detect samples at concentrations around 104 UCF per mL. The both methods, conventional (filtration technique) and alternative were done in parallel and the result data were used in the validation study. It was used in the assay the next parenteral solutions: physiological solution 0,9%, metronidazole solution 0,5%, dextrose solution 5% and Ringer lactate. The test solutions were inoculated with the microorganism suspensions, prepared by the dilution of the Bioballs® with result concentrations of 10 CFU/100 mL, 2 FU/100 mL and 0,4 CFU/ mL. After the conventional test was performed, the result membranes were incubated in thioglicollate and soybean casein broth. After 96 hours of incubation, aliquots from the broth were taken to perform the analysis by the alternative method. The following microorganisms were selected to perform the validation study: Staphylococcus aureus, Bacilus subtilis, Pseudomonas aeruginosa, Clostridium sporogenes, Candida albicans, Aspergillus brasiliensis, Kocuria rosea and Micrococcus luteus. The analysis of results shows that the alternative method can detect the test microorganisms. Regarding of the sensibility, the alternative method shows advantage in the 2 CFU/mL inoculum concentration and equivalence in the other two concentrations. The method shows evidence of robustness because the results were not affected by the products or culture media used in the assay. Additionally concerning the detection time, the alternative method was established equivalent to the conventional method (p-value=0,43).