Ideally, every person in the world should have access to a safe and clean water supply; if not all sources of water are clean and safe, at the very least, an effective method to detect water contamination should be readily available. An effective detection method should not only be sensitive, rapid, robust, and affordable, but, ideally, it should also be equipment-free and easy to transport and deliver to the end-users. The main goal of this project is to develop a variety of bits and pieces of bioassay systems, with a particular focus on paper-based bioactive devices in order to provide portable and ready-to-use biosensors which can be useable by anyone anywhere around the world without requiring formal training. According to the World Health Organization (WHO), 76,000 people each year die in India alone because of pesticide poisoning. Long term exposure to organophosphate pesticides is known to have adverse effects on neurological function and can lead to Alzheimer's Disease, Attention Deficit Hyperactivity Disorder (ADHD), and reduced Intelligence Quotient (IQ). The likelihood of long term exposure to pesticides is heightened in developing countries, so a reliable and inexpensive pesticide sensor is a much-needed device in the developing world. To address this need, this project reports on the development of a fully-automated bioactive paper-based sensor for the detection of organophosphate pesticides. In the proposed biosensor, two innovations were implemented to achieve a full-automated format for the pesticide sensor: (I) First is a PUMP ON A PAPER (Jahanshahi-Anbuhi et al., LOC, 2012) that increases the flow rate of fluids within paper-based microfluidic analytical devices and sequentially brings two separate liquid streams to the enzyme test zone on the paper sensor, and (II) the second innovation is a PIPETTE ON A PAPER (Jahanshahi-Anbuhi et al., LOC, 2014) that involved the creation of a pullulan (a natural non-ionic polysaccharide) temporary bridge-system to transfer a known amount of solution to the sensing zone that, gives the enzyme zone a chance to dry and accept the substrate solution from the slow channel after a fixed period of time. This proposed format results in a simplified assay that detects the presence of pesticides automatically without any further manipulation from the user. However, the shelf life of this assay kit is challenging due to instability of both enzyme (AChE) and substrate (IDA) at room temperature. AChE loses its enzymatic activity when stored at room temperature and IDA becomes oxidized quickly. This problem is not unique to these two bio reagents, however; almost all bioassays which use bio-reagents (such as enzymes and small-molecular substrates) are unstable to varying degrees and require special shipping and storage. The instability of these molecules can arise from either thermal denaturation or chemical modification, such as oxidation or hydrolysis. Because of these issues, they often have to be shipped on dry ice with special packaging, which is costly. The cost of maintaining a cold chain for distributing bio-reagents accounts for up to 80% of the cost. Aside from the cost, these reagents also have to be stored in bulk in refrigerators or freezers to minimize the loss of activity, but they must be thawed and aliquoted for their intended tests. Repeated freezing and thawing can result in a significant loss of activity, which often leads to less reliable test results. These issues make running such assays in resource-limited settings a significant challenge. There is, therefore, an urgent need for an assay system with stable reagents that is easy to use, simple to read, inexpensive, and that includes a method for the long-term stabilization of enzymes and other unstable reagents in pre-measured quantities. To overcome to all these issues, pullulan is utilized for the development of pill-based-biosensors. Pullulan dissolves quickly in aqueous solutions and shows very high oxygen barrier properties in its film form. Considering the unique properties of pullulan, it is hypothesized that pullulan may be suitable for producing assay pills with encapsulated enzymes or other unstable molecules and may provide a simplified platform for carrying out bioassays in resource-limited settings. The application of these pill-based-biosensors is shown via the entrapment of AChE and IDA for the creation of an assay kit that can detect organophosphate pesticides (Jahanshahi-Anbuhi et al., Angew. Chem., 2014). Moreover, this thesis reports on the stabilization of highly unstable firefly luciferase for the detection of microorganisms and, more particularly, ATP. Through the use of pullulan, this thesis demonstrates that both the enzyme and the substrate can be protected, immobilized, and stabilized at room temperature, instead of the existing storage methods, which require temperatures <-20˚C. This innovation allows for a more convenient method of shipping the bioassay kits around the world without any extra care. Furthermore, pullulan-based films are utilized for the development of a method for controlled multidirectional flow within paper-based biosensors. This method provides the possibility of trapping labile and volatile reagents and stabilizing them by forming thin films with pullulan. The trapped reagents within pullulan films can be strategically stacked and assembled on a paper strip in different directions. Furthermore, should the need arise, these reagents can be released and delivered sequentially or simultaneously in both vertical and lateral directions through the paper. The application of this method is shown for: (I) creation of "ready-to-use" assay kit for the detection of Escherichia coli (E. Coli). This assay kit has the step of cell lysing and proceeds automatically to the step in which enzymes react. The second application (II) shows the trapping of Simon’s reagents, which is widely used for methamphetamine detection. Overall, these unique fabrication techniques can be widely used for the preparation of highly stable, ready-to-use, and user-friendly biosensors. We are currently working on the detection of other contaminants such as heavy metals, and we are starting on vaccine stabilization and delivery, which would have a tremendous impact for society. / Dissertation / Doctor of Engineering (DEng)

No description available.

<p>Streptomycetes biology and genetics encompasses a variety of interesting features including multicellular growth, rich secondary metabolite production, and extensive environmental sensory and response systems. The characteristically large genomes of streptomycetes makes studying the diverse external stimuli intricate and internal regulation of these gene systems a challenge. Currently, there does not exist an efficient, cost-effective method of high throughput gene expression analysis in streptomycetes. Luciferase reporters have been used successfully in <em>Streptomyces coelicolor</em> to measure select promoter activity, however, they have demonstrated limited success in other strains and are not favourable to gene expression studies on a larger scale. Here, I present pLHR, a novel luciferase-based reporter tool designed specifically for high throughput gene expression studies in streptomycetes as well as the preliminary results which support the further development of this tool for gene expression profiling in <em>S. coelicolor</em>. Once developed, pLHR may be used to generate libraries of <em>Streptomyces</em> reporter stains to measure promoter activity repeatedly under variable conditions for the duration of the organism’s complex lifecycle.</p> / Master of Science (MSc)

Background Novel theranostic options for high-risk non-muscle invasive bladder cancer are urgently needed. This requires a thorough evaluation of experimental approaches in animal models best possibly reflecting human disease before entering clinical studies. Although several bladder cancer xenograft models were used in the literature, the establishment of an orthotopic bladder cancer model in mice remains challenging. Methods Luciferase-transduced UM-UC-3LUCK1 bladder cancer cells were instilled transurethrally via 24G permanent venous catheters into athymic NMRI and BALB/c nude mice as well as into SCID-beige mice. Besides the mouse strain, the pretreatment of the bladder wall (trypsin or poly-L-lysine), tumor cell count (0.5 × 106–5.0 × 106) and tumor cell dwell time in the murine bladder (30 min – 2 h) were varied. Tumors were morphologically and functionally visualized using bioluminescence imaging (BLI), magnetic resonance imaging (MRI), and positron emission tomography (PET). Results Immunodeficiency of the mouse strains was the most important factor influencing cancer cell engraftment, whereas modifying cell count and instillation time allowed fine-tuning of the BLI signal start and duration – both representing the possible treatment period for the evaluation of new therapeutics. Best orthotopic tumor growth was achieved by transurethral instillation of 1.0 × 106 UM-UC-3LUCK1 bladder cancer cells into SCID-beige mice for 2 h after bladder pretreatment with poly-L-lysine. A pilot PET experiment using 68Ga-cetuximab as transurethrally administered radiotracer revealed functional expression of epidermal growth factor receptor as representative molecular characteristic of engrafted cancer cells in the bladder. Conclusions With the optimized protocol in SCID-beige mice an applicable and reliable model of high-risk non-muscle invasive bladder cancer for the development of novel theranostic approaches was established.

Background Novel theranostic options for high-risk non-muscle invasive bladder cancer are urgently needed. This requires a thorough evaluation of experimental approaches in animal models best possibly reflecting human disease before entering clinical studies. Although several bladder cancer xenograft models were used in the literature, the establishment of an orthotopic bladder cancer model in mice remains challenging. Methods Luciferase-transduced UM-UC-3LUCK1 bladder cancer cells were instilled transurethrally via 24G permanent venous catheters into athymic NMRI and BALB/c nude mice as well as into SCID-beige mice. Besides the mouse strain, the pretreatment of the bladder wall (trypsin or poly-L-lysine), tumor cell count (0.5 × 106–5.0 × 106) and tumor cell dwell time in the murine bladder (30 min – 2 h) were varied. Tumors were morphologically and functionally visualized using bioluminescence imaging (BLI), magnetic resonance imaging (MRI), and positron emission tomography (PET). Results Immunodeficiency of the mouse strains was the most important factor influencing cancer cell engraftment, whereas modifying cell count and instillation time allowed fine-tuning of the BLI signal start and duration – both representing the possible treatment period for the evaluation of new therapeutics. Best orthotopic tumor growth was achieved by transurethral instillation of 1.0 × 106 UM-UC-3LUCK1 bladder cancer cells into SCID-beige mice for 2 h after bladder pretreatment with poly-L-lysine. A pilot PET experiment using 68Ga-cetuximab as transurethrally administered radiotracer revealed functional expression of epidermal growth factor receptor as representative molecular characteristic of engrafted cancer cells in the bladder. Conclusions With the optimized protocol in SCID-beige mice an applicable and reliable model of high-risk non-muscle invasive bladder cancer for the development of novel theranostic approaches was established.

Malaria remains one of the world's major health problems, especially in developing countries. A better understanding of the pathology and pathophysiology of severe malaria is key to develop new treatments. Different approaches have been used in malaria research including the in vitro co-culture models with endothelial cells and both murine and simian animal models. However these are open to controversy due to disagreement on their representativeness of human disease. Using human post-mortem tissue in malaria research is another important approach but is practically challenging, limiting the availability of post mortem samples from malaria patients. The work in this thesis had two main themes. First I examined the role of the endothelial signalling Angiopoetin-Tie-2 receptor pathway in malaria. Ang-2 has been shown to be a significant biomarker of severe and fatal malaria. I examined the tissue specific expression of proteins from this pathway in post-mortem brain tissues from fatal malaria cases, but found no difference between cerebral malaria and non-cerebral malaria cases. Ang-2 correlated with the severity of malaria in these patients. An attempt to examine the interaction of hypoxia and the Ang-Tie-2 pathway in vitro using a co-culture model of human brain endothelial cells was unsuccessful due to contamination of the cell line. The second part of the thesis aimed to utilise molecular pathology techniques including miRNA and whole-genome microarrays. I have shown for the first time that these can be successfully applied to human post-mortem tissue in malaria. First I used archival tissues to examine the microRNA signature in the kidney of patients with malaria associated renal failure. Second I optimised a protocol to preserve post mortem tissue for molecular pathology, from an autopsy study in Mozambique. Using the subsequent total mRNA transcriptomic data and bioinformatics analysis this work has expanded our knowledge of differential gene expression and the families of genes which are dysregulated in the brain in response to malaria infection.

Das Schilddrüsenhormon Triiodthyronin (T3) ist ein essenzieller Regulator physiologischer Prozesse der Entwicklung, des Wachstums und im Intermediärstoffwechsel. Täglich werden zahlreiche natürliche und synthetische Stoffe aufgenommen, die mit dem endokrinen System interferieren und deshalb als Endokrine Disruptoren (ED) bezeichnet werden. Zur Untersuchung einer direkten Interferenz von ED mit den Schilddrüsenhormonrezeptoren (TR) und ihrer transkriptionellen Aktivität wurde im Rahmen dieser Arbeit ein neues Luziferase-basiertes T3 Reportergensystem mit TRalpha1-transfizierten humanen Leberzellen aufgebaut. Durch Validierung mit dem synthetischen TR-Agonisten GC-1 und dem Antagonisten NH-3 konnte nachgewiesen werden, dass dieser Assay ein hoch-sensitives System zur Analyse von agonistischen sowie antagonistischen Effekten von Testsubtanzen darstellt. Zur Bestimmung der endokrinen Aktivitäten einiger humanrelevanter Vertreter aus den Stoffkategorien der Nahrungsmittel, Kosmetika, Pestizide und Industriechemikalien wurden Dosis-Wirkungskurven in Aktivierungs- und T3-Kompetitionsexperimenten ermittelt. In mikromolaren Konzentrationen wirkten von insgesamt 21 Testsubstanzen einige als reine Agonisten oder Antagonisten während andere gemischt agonistische/antagonistische Effekte hatten. Aufgrund ihrer hier beobachteten Effekte und der gegebenen Humanexposition wird eine eingehendere Analyse von 4-Methylbenzyliden Campher, 4-Nonylphenol, Acetochlor, Benzophenon 2, Benzophenon 3, Bisphenol A, Genistein, Octylmethoxycinnamat, Tetrabromobisphenol A und Xanthohumol empfohlen. Außerdem erwiesen sich einige Metaboliten von Schilddrüsenhormonen als potente Agonisten im T3-Reportergenassay und bedürfen weiterer Aufmerksamkeit. Für die molekulare Charakterisierung der Einflüsse solcher Substanzen auf die T3-regulierte Transaktivierung konnte mit dem hier etablierten Bioassay ein zuverlässiges neues Testsystem für reproduzierbare Screeningserien geschaffen werden. / Triiodothyronine (T3) is a crucial regulator of many physiological processes during development, growth and metabolism. A variety of natural and synthetic substances, which are collectively termed endocrine disrupters (ED) due to their interference with the endocrine system, is taken up on a daily base. A novel luciferase-based T3-responsive reporter gene system employing a human liver cell line transfected with thyroid hormone receptor (TR) alpha1 was established in this work to elucidate the potential molecular interference of certain ED with TR and their transcriptional activity. This assay was validated to be a highly sensitive and reliable tool for analyzing agonistic and antagonistic effects of test compounds using the synthetic TR agonist GC-1 and the antagonist NH-3. Dose-response data of test compounds contained in food, cosmetics, pesticides, plasticizers and other industrial chemicals were obtained after applying the substances alone in activation assays or in combination with T3 in competition assays. In total 21 test compounds were screened of which some acted as pure agonists or antagonists while others were mixed agonists/antagonists in the micromolar concentration range and only one was without effect. Follow-up studies are recommended for some of these substances with regard to their effects as determined in this bioassay and in light of information known on human exposure, i.e., 4-methylbenzyliden camphor, 4-nonylphenol, acetochlor, benzophenone 2, benzophenone 3, bisphenol A, genistein, octylmethoxycinnamate, tetrabromobisphenol A and xanthohumol. In addition some endogenous metabolites of thyroid hormones were surprisingly potent agonists in the T3 reporter gene assay and merit further attention. The novel bioassay established here represents a reliable tool for the screening and molecular characterization of substances interfering with T3-mediated transactivation of gene expression.

Au cours de cette thèse, deux sujets distincts ont été étudiés : d’une part la synthèse d’analogues de la coelentérazine, substrat de différentes luciférases, pour une application en imagerie in vivo, et d’autre part la synthèse d’analogues du (-)-EGCG, inhibiteur de la kinase Dyrk1a, impliquée notamment dans les troubles cognitifs rencontrés dans la trisomie 21.La problématique du premier sujet consistait à obtenir des analogues de la coelentérazine conservant leur activité sur deux luciférases, la luciférase Renilla et l’aequorine, tout en induisant un déplacement vers le rouge de la bioluminescence produite par ces enzymes. L’aequorine, sensible au calcium, représentait la cible biologique principale du projet.Sept analogues dont six originaux ont été obtenues par des méthodes de synthèse classiques, et leurs activités ont pu être testées sur les deux luciférases choisies, avec des résultats probants : malgré des émissions de lumière moins intenses que celles obtenues avec la coelentérazine native, plusieurs molécules ont entrainé un déplacement vers le rouge de la longueur d’onde d’émission de lumière par bioluminescence allant jusqu’à 27 nm pour l’aequorine et plus de 120 nm pour la luciférase Renilla.Le second sujet, de chimie médicinale classique, a principalement consisté à la synthèse d’analogues du gallate d’épigallocatéchine (EGCG) au squelette simplifié, et au sein desquels le cycle pyranique caractéristique des catéchines a été remplacé par un carbocycle. Plusieurs molécules ont pu être synthétisées, dont deux présentant le motif hexaphénol. Leur activité inhibitrice de Dyrk1a a pu être testée in vitro et l’une d’entre elle s’est déjà révélée plus active que l’EGCG. / During this thesis, two distinct projects were studied: on the one hand, the synthesis of coelenterazine analogs, substrate of several luciferases, in the purpose of using them for in vivo imaging, and on the other, the synthesis of (-)-EGCG analogs, inhibitor of the Dyrk1a kinase, which interests us for the role it plays in the mental retardation existing in the Down Syndrome disease.The problematic of the first project consisted in obtaining coelenterazine analogs that would not only maintain their activity on two luciferases, the Renilla luciferase and aequorine, but they should also induce a red-shift of the bioluminescence produced by these enzymes. Because of its sensitivity to calcium, aequorine was the main biologic target of this project.Seven analogs, of which six had an original structure, were synthesized through usual synthetic methodologies and their activities on both aequorine and Renilla luciferase were tested in vitro, with interesting results: even if the intensities of light emission were weaker than those obtained with native coelenterazine, several molecules produced a red-shift of the emission wavelength of bioluminescence, up to 27nm for aequorine and more than 120nm for the Renilla luciferase. The second project, of classical medicinal chemistry, mainly consisted in the synthesis of epigallocatechin gallate analogs (EGCG) with a simplified backbone and in which the pyranic ring typical of catechins was replaced by a carbocycle. Several molecules were synthesized, two of them possessing the hexaphenol motif. Their inhibiting activity of Dyrk1a was tested in vitro and one already showed a better activity than natural EGCG.

Large numbers of samples often need to be prepared and analysed in surveys of organic pollutants in the environment, but while the methods commonly used in such surveys can provide abundant detail they are generally costly, time-consuming and require large amounts of resources, so there is a need for simpler techniques. The work underlying this thesis assessed the potential utility of more convenient sample preparation and bioanalytical techniques for rapidly screening various environmental matrices that could be useful complements to higher resolution methods. Initially, the utility of a simplified extraction technique followed by an enzyme-linked immunosorbent assay (ELISA) for detecting polycyclic aromatic hydrocarbons (PAHs) in authentic (i.e. unspiked) contaminated soils was explored. The results showed that there are relationships between the structure and cross-reactivity among compounds that often co-occur with target PAHs. However, their potential contribution to deviations between estimates of total PAH contents of soils obtained using ELISA and gas chromatography-mass spectrometry (GC-MS) based reference methods were limited. Instead, the cross-reactivity of target PAHs and the failure to extract all of the PAHs prior to the ELISA determinations were the main reasons for these deviations. Polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) were detected in food and feed matrices, as well as in authentic contaminated soils using different bioanalytical techniques – ELISA and two cell-based bioassays: CAFLUX and CALUX (chemically activated fluorescent/luciferase gene expression) assays. In addition, enhanced sample preparation techniques based on accelerated solvent extraction (ASE) were developed. ASE with integrated carbon fractionation (ASE-C) in combination with CAFLUX produced estimates of PCDD and PCDF contents in fish oil and fish meal that agreed well with results obtained using reference methods. Furthermore, results from ELISA and GC-high resolution MS analyses of extracts of PCDD- and PCDF-contaminated soil samples obtained using an adjusted ASE-C technique were strongly correlated. Finally, the thesis reports the first experiments in which the results of CAFLUX, CALUX, and ELISA determinations of PCDDs and PCDFs in extracts of authentic contaminated soil samples were evaluated and compared to those obtained using a reference method. All of the bioanalytical techniques were found to be sufficiently sensitive, selective, and accurate for use in screening in compliance with soil quality assessment criteria. Overall, the improved sample preparation and bioanalytical techniques examined proved to be useful potential complements to conventional methods, enhancing the analytical framework for PAHs, PCDDs, and PCDFs. However, further validation has to be undertaken before they are applied on a large-scale.

The TRIB1 locus (8q24.13) is a novel locus associated with plasma TGs and CAD risk. Trib1 is a regulator of MAPK activity, and has been shown to regulate hepatic lipogenesis and VLDL production in mice. However, the functional relationship between common SNPs at the TRIB1 locus and plasma lipid traits is unknown; TRIB1 has not been identified as an eQTL. This cluster of SNPs falls within an intergenic region 25kb to 50kb downstream of the TRIB1 coding region. By phylogenetic footprinting analysis and DNA genotyping, we identified an evolutionarily conserved region (CNS1) within the risk locus that harbours two common SNPs in tight LD with GWAS risk SNPs and significantly associated with CAD. We investigated the regulatory function of CNS1 by luciferase reporter assays in HepG2 cells and demonstrate that this region has promoter activity. In addition, the rs2001844 risk allele significantly reduces luciferase activity, suggesting that altered expression of the EST-based gene may be associated with plasma TGs. We identified an EST within the risk locus directly downstream of CNS1. We performed 5'/3' RACE using HepG2 RNA, identified multiple variants of this EST-based gene, and confirmed its transcription start site within CNS1. We hypothesize that this EST is a long noncoding RNA due to low abundance, poor conservation, and absence of significant ORF. Over-expression of a short variant implicates its function in the regulation of target gene transcription, although the mechanism of action remains unknown. We conclude that the risk locus at 8q24.13 harbours a novel EST-based gene that may explain the relationship between GWAS SNPs at this locus and plasma lipid traits.