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1
Perry, John David. "Microbial growth indicators for the rapid assay of antimicrobial susceptibility." Thesis, Northumbria University, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.245287.
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Braddick, Darren. "Quantitative assay methods and mathematical modelling of peptidoglycan transglycosylation." Thesis, University of Warwick, 2012. http://wrap.warwick.ac.uk/57211/.
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The proportion of antibiotic resistant Gram-positive strains in the clinic and community continue to rise, despite the number of new antibiotics continuing to fall with time. At the intersection of this problem is the established challenge of working with what has ultimately been both nature’s and humanity’s favoured and most successful antibiotic target, the biosynthesis of the bacterial cell wall. The challenge lies in the predominately membrane/lipid linked habitat that the enzymes and substrates of this complex biosynthetic pathway function within. Membrane protein science remains non-trivial and often difficult, and as such remains undeveloped despite its hugely important role in the medical and biological sciences. As a result, there is a paucity of understanding for this pathway, with limited methods for assay of the activity of the biosynthetic enzymes. These enzyme include the monofunctional transglycosylases, monofunctional transpeptidase penicillin-binding proteins (PBPs) and bifunctional PBPs capable of both transglycosylation and transpeptidation. A number of these enzymes were expressed and purified, with the intention of obtaining novel kinetic and catalytic characterisation of their activities. The more complex of these enzymes could not be proven to be active, and so the comparatively simpler enzyme, an S. aureus monofunctional transglycosylase called MGT, was taken as a model enzyme and used to help design novel assay methods for its transglycosylase activity. The assays developed in this work gave access to novel time-course data and will help demonstrate other interesting mechanistic/catalytic information about the MGT enzyme and of transglycosylation in general. Mathematical modelling was performed around the experimental work. Novel and unique models were designed to define the mechanism of the MGT and generic transglycosylation, as this had not been performed before. The mathematical concepts of structural identifiability and structural indistinguishability were used to analyse these models. Data from experiments were then used to attempt data fitting with the models, and information about the underlying unknown kinetic parameters were collected. Together, a new framework of understanding of the MGT and transglycosylation can be made, which may hopefully be a small step towards answering the challenge now posed by widespread antibiotic resistance.
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Hurlburt, Allison L. "Molecular Padlock Assay of Crude Plant Leaf Extracts for Detection of Listeria Monocytogenes." Fogler Library, University of Maine, 2003. http://www.library.umaine.edu/theses/pdf/HurlburtAL2003.pdf.
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Zafer, Ahmed Abu. "Mycobactericidal testing of chemical germicides, comparison of conventional culture with a reporter gene assay." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape7/PQDD_0019/MQ48191.pdf.
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Cregger, S., D. Davis, Phillip R. Scheuerman, and M. Gallagher. "Development of a Macrophage Phagycytosis Assay for Immunotoxicolgy." Digital Commons @ East Tennessee State University, 1990. https://dc.etsu.edu/etsu-works/2888.
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Desjardins, Marc. "Feasibility of vaccination for chancroid: Sero-immunology and virulence assay in an experimental model of infection." Thesis, University of Ottawa (Canada), 1996. http://hdl.handle.net/10393/9969.
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Because of the well established epidemiologic and biologic interactions between human immunodeficiency virus (HIV) transmission and genital ulcer disease, measures to control chancroid could have a significant impact on the epidemic of HIV. I developed an IgG and IgM antibody serologic enzyme linked immunosorbent assay (ELISA) using pooled sera from clinically and microbiologically proven cases of chancroid as positive controls, and pooled sera from normal individuals without a history of sexually transmitted diseases as negative controls. Cross reactivity was minimized by absorption of serum samples with a sorbent prepared from three other Haemophilus species. Performance of the ELISA was enhanced in the period of early convalescence from acute primary chancroid and was not diminished in the presence of HIV coinfection, reflecting the tempo of the natural serologic response to and acute infection. I also developed an inhibition ELISA to determine antigenicity of potential vaccine candidates in human H. ducreyi infection, using lipooligosaccharide (LOS) as the test antigen. In a panel of 10 sera from cases of primary natural H. ducreyi infection reactive to H. ducreyi 35000 soluble antigen, only 4 samples were identified with reaction to H. ducreyi 35000 LOS. Inhibition ELISA confirmed that pre-adsorption with LOS substantially diminished reactivity of these sera to LOS but not to soluble antigen. Using the ELISA to detect immunogenicity by measuring the serologic response to vaccine candidates in rabbits, we further tested the feasibility of three bacterial antigen preparations to induce protective immunity against infection and disease. LOS carbohydrate and a pilus preparation were purified from H. ducreyi 35000 and used in a booster immunization procedure. Virulence was assayed by intra-epithelial challenge and measurement of disease for homologous strain 35000 or the virulent clinical isolate RO-34. LOS and the pilus preparation both induced humoral responses to the corresponding antigen, but the carbohydrate preparation did not. Immunization with LOS or carbohydrate did not modify virulence of infection with H. ducreyi 35000. Immunization with the strain 35000 pilus preparation significantly reduced the severity of disease, and the duration of infection and disease compared with controls. I conducted passive immunization experiments, and characterized the inflammatory infiltrate of chancroidal lesions. Naive rabbits were passively immunized with 24 or 48 mg of purified polyclonal IgG intravenously and 24 hours after infusion, challenged with the homologous strain 35000. No significant difference in disease resulting from infection with the homologous strain was observed. I then comparatively evaluated the serial immunohistology of lesions produced by infectious challenge with the homologous strain in sham-immunized or rabbits immunized with the pilus preparation. Flow cytometric analysis of rabbit peripheral blood leucocytes with CD5 and CD4 rabbit lymphocyte markers prior to infectious challenge revealed two T lymphocyte populations: CD5+CD4+ and CD5+CD4$-$. Pilus preparation vaccinee lesions showed significant quantitative acceleration and increase in T lymphocyte infiltration, and similar early increased recruitment of the CD4+ T lymphocyte subset preceding early lesion sterilization and early healing without ulceration. (Abstract shortened by UMI.)
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Sandhu, Sandeep. "Development of an antibody-based assay for methicillin resistant Staphylococcus aureus using synthetic peptidoglycan precursors." Thesis, University of Warwick, 2010. http://wrap.warwick.ac.uk/36857/.
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Methicillin-resistant Staphylococcus aureus (MRSA) are isolates of the bacterium Staphylococcus aureus that have acquired genes encoding antibiotic resistance to all penicillins, including methicillin and other narrow-spectrum β- lactamase-resistant penicillin antibiotics. Outbreaks of MRSA occur quite frequently as there is no quick screening test for the presence of MRSA. The aim of this project is to try and develop an antibody based detection test for rapid detection of MRSA, which could help in the prevention of outbreaks. An antigen (UDP-MurNAc-L-Ala-γ-D-Glu-L-Lys(ε-NH2-Gly)5-D-Ala-D-Ala) that resembles the outer surface of the Staphylococcus aureus (contains a Gly5 moiety that is specific for S. aureus) cell wall peptidoglycan has been prepared, and attached to a carrier protein. Sheep antibodies raised against this antigen were screened using ELISA assays. The results showed that antibodies did have specificity for the antigen. Cell walls were prepared from several different bacteria, including two MRSA and one methicillin-sensitive Staphylococcus aureus (MSSA) strain. ELISA assays using these cell walls showed that the antibodies had specificity for cell walls containing (Gly)5 in the order of S aureus (Gly5) > S. simulans (less Gly5) > S. pneumoniae (no Glyn) > E. coli (no Glyn, Gram-negative strain). A particularly high response was observed for one of the two MRSA strains, detectable at 0.1 μg of cell wall. An HPLC-based UV-Vis assay was developed to monitor the activity of peptidoglycan polymerisation enzymes from S. aureus, while preparing polymeric peptidoglycan as an antigen for immunisation. Several intermediates in peptidoglycan polymerisation were detected using S. aureus monofunctional glycosyltransferase (MGT), which allowed us to propose a new hypothesis for the early steps of peptidoglycan transglycosylation.
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Meloche, Michèle. "Development and application of a quantitative virulence assay for Haemophilus ducreyi in an in vivo model of infection." Thesis, University of Ottawa (Canada), 1993. http://hdl.handle.net/10393/10955.
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A temperature-dependant rabbit model of Haemophilus ducreyi infection was used as a quantitative virulence assay to evaluate the effect of host factors upon ulcerative cutaneous disease production. New Zealand White rabbits underwent inoculation with H. ducreyi strain #35000 either after iron loading, dexamethasone immunosuppression, prior infection or immunization. Chancroid-like disease was scored for severity, time, culture of lesions, and serologic response. In primary infections, culture-positive ulcerative lesions were consistently produced at and above 105 colony forming units inocula. Iron and dexamethasone treatment increased lesion severity and duration of culture positivity. Infection of previously infected animals produced sterile lesions of greater size and higher cumulative disease score at 105 colony forming units inocula. Infection of immunized animals produced sterile lesions of lesser severity at 105 colony forming units, with protection from ulcer formation. Efficacy of ceftriaxone treatment was tested in naive control and iron loaded rabbits. Antibiotic was injected as a single intramuscular dose of 0.1 mg/Kg and 5 mg/Kg, four days following inoculation with Haemophilus ducreyi #35000. In naive control rabbits, antibiotic treatment at each dose sterilized the lesions within 24 hours with attenuation of disease effect. In naive iron loaded rabbits lesions were sterilized later on day 10 with 0.1 mg/Kg ceftriaxone and on day 5 with 5 mg/Kg ceftriaxone. Virulence scores corroborated the lessened microbiologic efficacy of antibiotic treatment. We conclude that quantitative assay of infection and disease in iron loaded animals, with relative prolongation of disease effect and culture positivity of lesions may be a sensitive model in which to comparatively measure virulence related to bacterial factors. As well, limitations of efficacy or synergy of antibiotic treatments may be evaluated. We conclude that prior antigenic exposure and immune response through infection or immunization attenuates subsequent homologous infection. Disease produced in re-infection is amplified, while disease produced in inoculation after immunization is attenuated at lower inocula. This suggests that while there is inducible immunity to H. ducreyi infection and disease, the disease effect may be in part related to host inflammatory response to bacterial antigen or effect of bacterial toxin. This system of measurement of virulence may be useful towards understanding pathogenesis, and identifying strategy for vaccine development in chancroid.
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Hill, S. A. "An evaluation of potato virus Y and potato leaf roll virus detection in tubers by enzyme-linked immunosorbent assay." Thesis, University of Reading, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.370134.
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Burns, Anthony J. "Gut lumen factors, including probiotics and prebiotics, influencing faecal water genotoxicity on HT29 cell line using the comet assay." Thesis, University of Ulster, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.232843.
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O'Reilly, Amy. "Assay development towards the characterisation of the bifunctional activity of P. aeruginosa and E. coli Class A PBPs." Thesis, University of Warwick, 2017. http://wrap.warwick.ac.uk/93414/.
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Pseudomonas aeruginosa is a Gram-negative species of bacteria that is of high clinical importance and is on the ESKAPE list of pathogens, causing the most serious nosocomial infections World-wide. Current antibiotics in clinical use against P. aeruginosa include carbenicillin, cefepime, ceftazidime, and the fluoroquinoline ciprofloxacin. The multi-drug resistant (MDR) phenotype exhibits widespread resistance to β-lactams and antibiotics from multiple classes often need to be administered in conjunction with β-lactamase inhibitors. Penicillin binding proteins (PBPs) are essential for cell viability and are important antibiotic targets, with the Class A bifunctional PBPs synthesising the principle component of the bacterial cell wall, peptidoglycan. The focus of this project was to investigate the kinetics of transglycosylation and transpeptidation mechanisms with a view to improving our understanding of bacterial cell wall synthesis, maintenance and regeneration. In this project, the function of Class A PBPs 1A and 1B from Escherichia coli and P. aeruginosa, with particular focus on PBP1A has been investigated. Transglycosylation and transpeptidation have been probed from multiple angles using orthogonal assays and fluorescently-labelled and native lipid substrates, as well as Lipid II structural variants. P. aeruginosa PBP1A transglycosylase and transpeptidase activities are demonstrated continuously, and compared to the model organism equivalent, E. coli PBP1A. The continuous monitoring of activity gave insights into the possibility of two catalytic sites for transglycosylation: a donor and an acceptor site. Data was fitted to different kinetic models to elucidate the best fit for extracting meaningful kinetic parameters. The data acquired over the course of this project suggests that P. aeruginosa PBP1A and E. coli PBP1A exhibit similar functional behaviour to each other, with E. coli PBP1B showing distinct activity from PBP1A. PaPBP1B activity was not detected at a level required to kinetically characterise the enzyme and it is possible that this PBP has an as yet undetermined stimulatory cofactor. The assays developed and optimised in this thesis will pave the way for further in-depth studies of P. aeruginosa PBP1A, which could be used to screen for putative inhibitor compounds and potentially identify certain characteristics required for antimicrobial efficacy. Over the past 4 years, major leaps in our knowledge of assay development, the transglycosylase and transpeptidase activities of Class A PBPs and substrate features and requirements for transglycosylase and transpeptidase donors and acceptors have thus far and will continue to unravel the unique and fascinating mechanistic intricacies of the bifunctionality of PBPs.
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Hoppe, Kimberly Ann. "Bioaerosol exposure assessment and the Limulus amoebocyte lysate assay." Diss., University of Iowa, 2013. https://ir.uiowa.edu/etd/4858.
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In June 2008, the Cedar River crested flooding more than 5,000 Cedar Rapids homes. Residents whose homes were flooded were invited to participate in this study. We characterized exposures and symptoms experienced by individuals inhabiting 73 flood-damaged homes. Exposures and questionnaire-based health assessments were compared at two levels of remediation, in-progress and completed. Homes with remediation in-progress (n=24), as compared to the completed homes (n=49), had significantly higher airborne concentrations of mold, bacteria, iPM, endotoxin and glucan. Residents of in-progress homes had a significantly higher prevalence of doctor diagnosed allergies (adjusted OR=3.08; 95%CI: 1.05-9.02) and all residents had elevated prevalence of self-reported wheeze (adjusted OR=3.77; 95%CI: 2.06-6.92) and prescription medication use for breathing problems (adjusted OR=1.38; 95%CI: 1.01-1.88) after the flood as compared to before. Proper post-flood remediation led to improved air quality and lower exposures among residents living in flooded homes.
Recognition of endotoxin as a proinflammatory ligand for pattern recognition receptors has increased the demand for endotoxin assessment in studies of environmental lung disease. Measurements using the Limulus amebocyte lysate (LAL) assay of air and reservoir dust samples are routinely incorporated into epidemiologic studies. However, it is unknown if endotoxin reactivity in the LAL assay varies by its physical presentation as aggregates, as membrane components of whole bacteria or as shed membrane blebs or if this parallels differences in the inflammatory potency of endotoxin in vivo. Endotoxins as14C-labeled-lipooligosaccharide (14C-LOS) and 14C- labeled-lipopolysaccharide (14C-LPS) were produced from Neisseria meningitidis and Escherichia coli. The reactivity of the endotoxin presentations was assessed in the LAL assay and in vivo using a murine model. The LAL assay significantly underestimated the quantity of endotoxin in the whole bacteria form whereas there was no significant difference in detecting endotoxin in aggregate and bleb forms. The failure of the LAL assay to equally quantify endotoxin was not mirrored in vivo where all three presentations of endotoxin were equally inflammatory.
The inability of the LAL assay to detect the full quantity of endotoxin presented in the whole bacteria form has troubling implications for exposure assessment studies. Various extraction methods were applied to samples of known endotoxin quantity to improve the detection ability of the LAL assay. Extraction using EDTA and Tris/EDTA significantly improved the detection of endotoxin compared to the reference method of extracting in pyrogen-free water. These extraction methods also significantly increased the quantity of endotoxin measured in house and barn dust samples. A higher quantity of endotoxin measured in the LAL assay corresponded to a higher neutrophilic response in vivo. A standardized methodology for endotoxin detection that mimics the in vivo response is necessary for accurate and consistent endotoxin analysis.
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Botha, Elizabeth Magdelena. "Molecular characterization of South African lineage II West Nile virus isolates ltime PCR assay." Pretoria : [s.n.], 2008. http://upetd.up.ac.za/thesis/available/etd-06122008-130924/.
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MacRae, Jean Dorothy. "Characterization of Caulobacters isolated from wastewater treatment systems and assay development for their enumeration." Thesis, University of British Columbia, 1990. http://hdl.handle.net/2429/30112.
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Caulobacters are gram-negative bacteria that have a biphasic life cycle consisting of a swarmer and a stalked stage. As a result they have elicited interest as a simple developmental model. Less attention has focussed on their role in the environment, although they have been found in almost every aquatic environment as well as in many soils. Caulobacters are often described as oligotrophic bacteria because of their prevalence in pristine waters but have now been isolated from the relatively nutrient-rich wastewater environment. In order to learn more about this population some basic characterization was carried out and an assay system to determine their prevalence in sewage plants was designed.
Most of the organisms isolated from sewage treatment facilities had similar gross morphological features, but differed in holdfast composition, total protein profile, antibiotic resistance and restriction fragment length polymorphism, thereby indicating a greater diversity than originally assumed. Most of the organisms hybridized with flagellin and surface array genes that had previously been cloned, and only one of 155 non-Caulobacter sewage isolates hybridized with the flagellin gene probe; consequently these were used in a DNA-based enumeration strategy.
DNA was isolated directly from sewage and probed with the flagellin and the surface array gene probes. The signals obtained were compared to standards made up of pooled Caulobacter DNA from the sewage isolates and non-Caulobacter DNA from organisms also present in sewage. Using this assay Caulobacters could only be detected above the 1% level, which was higher than their proportion in the wastewater environment. It appears that this approach will not be useful in monitoring Caulobacters in treatment plants unless a more highly conserved or higher copy number probe is found.Science, Faculty of
Microbiology and Immunology, Department of
Graduate
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Segal, Jonathan. "Real-time qPCR Assay Development for Detection of Bacillus thuringiensis and Serratia marcescens DNA and the Influence of Complex Microbial Community DNA on Assay Sensitivity." FIU Digital Commons, 2013. http://digitalcommons.fiu.edu/etd/1030.
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Real-time quantitative polymerase chain reaction (real-time qPCR) assays are an effective technique to detect biological warfare agents and surrogate organisms. In my study, primers were designed to detect chromosomal DNA of biological warfare agent surrogates B. thuringiensis and S. marcescens (representing B. anthracis and Y. pestis, respectively) via real-time qPCR. Species-level specificity of the primers was demonstrated through comparisons with a bacterial strain panel and corroborated by qPCR data. Additionally, the primer efficacy was tested when template DNA was spiked into metagenomic DNA extracted from clinical lung microbiome samples. The results showed that while detection of B. thuringiensis or S. marcescens was still largely successful, the addition of metagenomic DNA did significantly inhibit amplification in most cases. The present study is significant not only for the design of multiple novel primer pairs able to detect bacterial agents in metagenomic DNA, but also the quantitative insight to the influence of background DNA on single species detection at low DNA concentrations.
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Ntuli, Sindile Venessa. "Validation of a Pan-fungal polymerase chain reaction (PCR) assay for the detection and identification of medically important fungi in a diagnostic laboratory." Master's thesis, University of Cape Town, 2018. http://hdl.handle.net/11427/27816.
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The laboratory diagnosis of fungal infection is complicated, based on the microscopic detection, culture isolation, and detection of serological response. In recent years, there has been an increase in the utilization of molecular diagnostic techniques for the detection and accurate identification of fungal pathogens. This was a laboratory accuracy study evaluating the performance of a selected pan-fungal PCR using 70 previously identified reference fungal isolates. The DNA yield and purity of three different DNA extraction methods was assessed, using 6 representative fungal isolates. The ZR Fungal/Bacterial DNA MicroPrep™ produced a median concentration of 17.28 ng/μl,which was significantly higher (p value = 0.0079) than the MagNA pure LC DNA Isolation Kit III and QIAamp DNA Mini Kit, which produced median yields of 11.08ng/μl and 3.54 ng/μl, respectively. The selected pan-fungal PCR was optimized PCR and successfully performed on 62 of the 70 reference isolates. A selection of 56 amplicons were submitted for DNA sequence determination. Of all the sequences queried on the NCBI and Ribosomal Development Project (RDP) databases, 95/111(86%) were concordant with the results obtained from the reference laboratory. Study findings have shown that the selected pan-fungal PCR, coupled with DNA sequence analysis is an excellent diagnostic tool for the identification of medically relevant fungi. This assay, in combination with conventional culture, is useful for the rapid and accurate identification of fungal isolates. Future work will involve evaluating the utility of this assay for the detection and identification of medically relevant fungi in deep tissue biopsies.
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Glascock, S. J. "A study of #Beta#-glucuronidase with emphasis on the development of a rapid quantifiable assay for Escherichia coli detection." Thesis, University of Lincoln, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.263962.
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Joshi, Lovleen Tina. "Pathogenicity & a bedside real-time detection assay for clostridium difficile in the faeces of hospitalized patients." Thesis, Cardiff University, 2012. http://orca.cf.ac.uk/37079/.
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Clostridium difficile, a Gram positive, anaerobic, spore-forming bacterium is the commonest cause of hospital acquired infection in the UK. The organism initiates infection through spore formation and attachment, germination in the gut and then the production of two potent cytotoxins; toxins A and B. While the contribution of toxins A and B to infection is beyond dispute the relative importance of each toxin is a subject of debate. Thus diagnostic assays capable of rapidly detecting the presence of both toxins are needed. To develop such an assay we first characterised the structure of C. difficile spores to better understand their role in pathogenicity and adherence to organic and inorganic surfaces. Following attachment the spore germinates and the resulting vegetative bacteria express toxins. To facilitate the development of an assay capable of detecting both toxins, we employed a bioinformatics based approach which identified highly conserved nucleotide sequences within regions of each toxin which we hypothesised were under strict selective pressure. The specificity of the probes identified was confirmed using a panel of 58 clinical C. difficile isolates, related Clostridium isolates, non-related species and human gut metagenomic DNA samples. Selected probes were incorporated into a metal enhanced fluorescent assay platform and their ability to detect the organism in various organic backgrounds was determined. We were able to detect as few as 10 bacteria in 500 μl of human faecal material within 40 seconds, suggesting that this approach has the potential to be developed into a commercial assay. To support the development of this assay we sought to develop an insect infection model using the worm Manduca sexta. Our inability to initiate infection, inspite of the fact that bioinformatic analysis revealed the presence of genes with homology to known insect virulence factors, suggests that C. difficile may have potential evolutionary association to invertebrates.
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Garrett, Andrew Robert. "Antioxidants in Cancer Research and Prevention: Assay Comparison, Structure-Function Analysis, and Food Product Analysis." BYU ScholarsArchive, 2011. https://scholarsarchive.byu.edu/etd/2735.
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Recent epidemiological studies have suggested that the development and progression of several chronic diseases may be initiated or augmented by oxidative stress. Reactive oxygen species and reactive nitrogen species react readily with and can damage nucleic acids, proteins, and lipids. While biological systems are equipped antioxidant defenses to cope with oxidative stress, oxidative damage may still occur when oxidative stress overwhelms antioxidant defenses. This damage, if left unchecked, may lead to a variety of degenerative diseases, including heart disease, Alzheimer's Disease, Parkinson's Disease and cancer. Several assays have been designed to describe the antioxidant activity of various phytochemicals, vitamins, and other compounds. The ORAC and TOSC assays have emerged as industry standards for measuring antioxidant activity due to their high reliability and sensitivity. Until recently, however, little has been done to assess the relative correlation between these two assays. Furthermore, no assay has been developed to measure changes in antioxidant activities of cells in response to oxidative stress. The current work investigates the correlation between measured antioxidant activities of samples in the both the ORAC and TOSC assays. Recent antioxidant research also focuses on relating chemical structure to antioxidant activity. Previous research in this area has included a broad range of chemical groups, but no study has attempted to formulate a structure-function framework that has applicability to compounds of any group. The current work uses amino acids as a simplest-case model for studying the relationships between chemical structure and antioxidant activity. One particular area of emerging research has centered around comparing organic and conventionally grown food products. The impetus of these investigations lies in claims made by organic supporting groups that these food products are generally more beneficial than their conventional counterparts. Despite the rapid rise in popularity of organic foods, there remains a dearth of research investigating these claims. The current work compares the antioxidant activities of organic and conventionally grown blueberries and apples.
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Gantelius, Jesper. "Novel diagnostic microarray assay formats towards comprehensive on-site analysis." Doctoral thesis, Stockholm : Skolan för bioteknologi, Kungliga Tekniska högskolan, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-11221.
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Margolin, Aaron B. 1958. "The development of a dot blot assay using gene probes for the detection of enteroviruses in water." Diss., The University of Arizona, 1986. http://hdl.handle.net/10150/191106.
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Enteric viruses are viruses which replicate in the intestinal tract of man and animals. One mode of transmission for enteric viruses is the fecal-oral route. Drinking water which has been contaminated with sewage or sewage effluent has been implicated as a means for the spread of enteric viruses. Monitoring water for virus contamination requires two steps: 1) the collection and the concentration of the water sample and 2) the isolation and identification of the virus present. Current methods for the detection of enteric viruses in water requires the use of animal cell culture. This technique has several drawbacks, such as: 1) long incubation periods, up to two and three weeks, before some enteric viruses are detected, 2) not all viruses can be detected in one cell line, and 3) not all viruses have been grown in cell culture. More rapid techniques, such as fluorescent antibody or radioimmunoassay do not have the needed sensitivity to detect the low levels of virus found in contaminated water. These techniques also require the production of an antibody for each different virus type. An alternative technique for the detection of viruses in water was sought. Recent advances in recombinant DNA technology now makes it possible to detect viruses without the use of cell culture or antibodies. Gene probes that hybridize to the RNA of poliovirus and hepatitis A virus were tested for their ability to detect different enteric viruses. The probes were labeled with ³²P dCTP and ³²P dATP to a specific activity greater then 1.0 x 10⁹ cpm/ug DNA. Gene Screen Plus (NEN) was chosen as the hybridization membrane since it was more sensitive to virus detection than the other membranes tested. A dot-blot apparatus (Bio Rad) was used to apply the samples. Results were visualized by autoradiography for thirty-six hours at -70° C. One infectious unit of poliovirus and hepatitis A virus was detected using labeled cDNA probes. Upon comparison, the dot blot assay was as sensitive as tissue culture for the detection of poliovirus in beef extract, secondary effluent, and tapwater. Environmental samples, such as secondary effluent, reclaimed wastewater and unchlorinated drinking water were also assayed for poliovirus and hepatitis A virus with the use of gene probes. The results presented here offer an alternative method for screening water samples for the presence of enteric viruses.
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Safodien, Sieyaam. "The molecular identification and characterisation of Eutypa dieback and a PCR-based assay for the detection of Eutypa and Botryosphaeriaceae species from grapevine in South Africa." Thesis, Stellenbosch : Stellenbosch University, 2007. http://hdl.handle.net/10019.1/21757.
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Thesis (MSc)--University of Stellenbosch, 2007.ENGLISH ABSTRACT: Grapevine trunk diseases are caused by invasive pathogens that are responsible for the slow decline of vines. In particular, Eutypa dieback of grapevine has had a devastating impact on vineyards worldwide, reducing growth and yield, eventually killing the grapevine. The causal organism of Eutypa dieback was first described as Eutypa armeniacae Hansf. & Carter, the pathogen that causes dieback of apricots, but since 1987 this species has been considered a synonym of Eutypa lata (Pers.:Fr.) Tul & C. Tul (anamorph Libertella blepharis A. L. Smith). Recently, it was proposed that at least two species that are capable of infecting grapevines are responsible for Eutypa dieback. Consequently, the molecular identification and characterisation of Eutypa dieback was used to delineate the species occurring on infected grapevines in South Africa. This involved the molecular analyses of three molecular markers, namely, the internal transcribed spacer (ITS) and large subunit (LSU) regions of the ribosomal DNA operon, and the -tubulin gene. The results obtained revealed the presence of a second species, namely, Eutypa leptoplaca (Mont.) Rappaz, that occurred together with E. lata on infected grapevines. Also co-habiting with these pathogens were related fungi form the Diatrypaceae family, Cryptovalsa ampelina (Nitschke) Fuckel and Eutypella vitis (Schwein.) Ellis & Everhart. Pathogenicity tests conducted on isolates representing C. ampelina, E. lata, E. leptoplaca, and E. vitis revealed that all were pathogenic to grapevine. Several species of Botryosphaeriaceae that commonly invade the woody tissue of grapevines are also pathogenic to grapevine. The symptoms in grapevine commonly associated with Botryosphaeriaceae are easily confused with the symptoms produced by Eutypa dieback which prompted the need for the development of a detection method that can correctly identify the presence of multiple pathogens. A reverse dot blot hybridisation (RDBH) method was subsequently applied to provide a rapid, accurate and reliable means of detecting the Eutypa species involved in the Eutypa disease complex, as well as those species of Botryosphaeriaceae known to cause disease in grapevines. The method involved the use of multiplex PCR to simultaneously amplify and label the regions of DNA that are used as pathogen specific probes. Consequently, membrane immobilised species-specific oligonucleotides synthesised from the ITS, - tubulin and LSU molecular data were evaluated during the application of this diagnostic method to detect Eutypa species. It was found that the species-specific oligonucleotides, designed from ITS sequence data, could consistently detect E. lata and E. leptoplaca. The application of the RDBH method for the detection of these Eutypa species, based on -tubulin and LSU sequence data, however, proved to be unsuccessful. Subsequently, a RDBH method, utilising species-specific oligonucleotides designed from elongation factor-1α sequence data, was successfully applied for the detection of Botyrosphaeria dothidea (Moug.:Fr.) Ces. & De Not., Neofusicoccum luteum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips, Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers, A.J.L. Phillips and Neofusicoccum ribis (Slippers, Crous & M.J. Wingf.) Crous, Slippers & A.J.L. Phillips. The method, however, was unsuccessful for the detection of Diplodia seriata De Not. In addition to the above-mentioned shortcomings, the RDBH was not amenable to the detection of pathogens directly from field or environmental samples, but required preparation of DNA from pure cultures. The method, however, allows for the identification of multiple pathogens in a single assay. As DNA extraction methods are amended, improved and honed to obtain DNA from environmental samples, so would it increase the usefulness of RDBH.
AFRIKAANSE OPSOMMING: Wingerd stamsiektes word veroorsaak deur patogene wat die vermoë het om wingerdplante te infekteer en dan stadige agteruitgang van dié wingerde te veroorsaak. Veral Eutypa terugsterwing het ‘n vernietigende effek op wingerde wêreldwyd deurdat dit groeikrag en oesmassa verlaag, maar ook omdat dit uiteindelik wingerdstokke kan dood. Die veroorsakende organisme is aanvanklik as Eutypa armeniacae Hansf. & Carter beskryf, die patogeen wat terugsterf by appelkose veroorsaak, maar sedert 1987 word hierdie spesies beskou as ‘n sinoniem van Eutypa lata (Pers.:Fr.) Tul & C. Tul (anamorph Libertella blepharis A. L. Smith). Dit is egter onlangs voorgestel dat ten minste twee spesies die vermoë het om wingerd te infekteer om Eutypa terugsterwing te veroorsaak. Gevolglik is molekulêre identifikasie- en karakteriseringstudies geloods om te bepaal watter spesies Eutypa terugsterwing in Suid-Afrikaanse wingerde veroorsaak. Dit het die molekulêre analise van drie molekulêre merkers behels, naamlik die interne getranskribeerde spasiëerderarea (“ITS”), die groot ribosomale subeenheid (“LSU rDNA”) en β-tubilien geen. Resultate van die filogenetiese analise dui daarop dat ’n tweede spesies, naamlik Eutypa leptoplaca (Mont.) Rappaz, saam met E. lata in geïnfekteerde plante voorkom. Saam met bogenoemde twee spesies het daar ook verwante spesies van die Diatrypaceae familie voorgekom, naamlik Cryptovalsa ampelina (Nitschke) Fuckel en Eutypella vitis (Schwein.) Ellis & Everhart. Patogenisiteitstudies wat uitgevoer is met verteenwoordigende isolate van C. ampelina, E. lata, E. leptoplaca, en E. vitis dui daarop dat almal patogene van wingerd is. Verskeie Botryosphaeriaceae spesies wat gereeld in houtagtige wingerdweefsel aangetref word, is ook patogene van wingerd. Interne simptome wat algemeen met Botryosphaeriaceae infeksies geassosieer word, kan baie maklik met dié van Eutypa terugsterwing verwar word en dit het die nood laat ontstaan om ‘n opsporingsmetode te ontwikkel wat akkuraat genoeg is om tussen veelvoudige infeksies te onderskei. ’n Omgekeerde-stippelklad-hibridisasie (OSH) metode is gevolglik aangewend om Eutypa spesies betrokke in die Eutypa-siektekompleks op ‘n vinnige, akkurate en betroubare manier op te spoor, sowel as die Botryosphaeriaceae species wat bekend is as patogene van wingerd. Die metode behels ’n saamgestelde PKR vir die vermeerdering en merk van DNS areas wat gebruik word as patogeen spesifieke peilers. Spesies-spesifieke oligonukleotiede ontwikkel vanaf die ITS, -tubilien en LSU molekulêre data is op ‘n membraan vasgeheg en gebruik om ’n diagnostiese toets te ontwikkel vir Eutypa species. Merkers ontwikkel vanaf die ITS kon E. lata and E. leptoplaca konsekwent opspoor. Die opspoor van Eutypa spesies met merkers vanaf die -tubulien en LSU gene met OSH was onsuksesvol. Die OSH metode met merkers vanaf die verlengingsfaktor-1α kon susksesvol gebruik word om Botyrosphaeria dothidea (Moug.:Fr.) Ces. & De Not., Neofusicoccum luteum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips, Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers, A.J.L. Phillips and Neofusicoccum ribis (Slippers, Crous & M.J. Wingf.) Crous, Slippers & A.J.L. Phillips op te spoor. Dié metode kon egter nie Diplodia seriata De Not. opspoor nie. Bykomend tot bogenoemde tekortkominge, kon die omgekeerde-stippelklad-hibridisasie metode ook nie aangepas word om patogene direk vanuit plantmateriaal op te spoor nie en word DNS afkomstig vanaf suiwer kulture benodig. Dié metode laat egter identifikasie van verskeie patogene in ‘n enkele toets toe. Soos DNS ekstraksie metodes aangepas, verbeter en verfyn word om DNS vanuit plantmateriaal te verkry, sal die bruikbaarheid van die omgekeerde stippelklad hibridisasie metode ook verbeter.
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Livingston, Justin Ryan. "The Antioxidant and DNA Repair Capacities of Resveratrol, Piceatannol, and Pterostilbene." BYU ScholarsArchive, 2015. https://scholarsarchive.byu.edu/etd/5525.
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Lifestyle diseases represent a large burden on developed societies and account for much morbidity worldwide. Research has shown that eating a diet rich in fruit and vegetables helps to ameliorate and prevent some of these diseases. Antioxidants found in fruits and vegetables may provide a substantial benefit in reducing disease incidence. This thesis examines the antioxidant properties of resveratrol, piceatannol, and pterostilbene, and the ability of Burkitt's Lymphoma (Raji) cells to uptake these three antioxidants. It also studies the effect of the antioxidants in protecting against DNA damage and their role in DNA repair following oxygen radical exposure in Raji cells. The Oxygen Radical Absorbance Capacity (ORAC) assay was used to measure overall antioxidant contribution as well as the ability of Raji cells to uptake antioxidant following exposure to 2,2’-Azobis(2-methyl-propionamide) dihydrochloride (AAPH). The single cell gel electrophoresis (Comet) assay was used to assess DNA damage and DNA repair rates of cells. Results showed that Raji cells, following oxygen radical exposure, significantly uptake pterostilbene (p < 0.0001), but not piceatannol or resveratrol. Piceatannol provided protection against hydrogen peroxide induced DNA damage, but pterostilbene and resveratrol increased DNA damage following hydrogen peroxide treatment. None of the compounds showed any effect on DNA repair. Overall, this study indicates there is merit for further research into the bioactive roles, including antioxidant capacity, of all three compounds. Such research may provide evidence for the more widespread use of these and other food based compounds for preventing lifestyle diseases.
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Wai, Chi-wan, and 衛至韻. "Development of shell vial culture assay for the rapid diagnosis of respiratory viruses using the human colorectal adenocarcinoma (CaCo2) cells." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2013. http://hdl.handle.net/10722/193551.
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Background: Respiratory diseases are common worldwide, which are caused by various respiratory viruses. As symptoms caused by these viruses are similar, laboratory diagnosis is essential to distinguish the virus. Conventionally, respiratory viruses are isolated by cell culture with a panel of cell lines. However, handling of several cell lines is labour intensive, and the turnaround time of conventional culture is long. In previous study, the use of human colon adeno-carcinoma (Caco-2) in conventional culture was investigated. The study has proven that Caco-2 is generally susceptible to the eight common respiratory viruses, i.e. Adenovirus, Influenza A and B, Respiratory Syncytial virus, Parainfluenza virus 1, 2,3 and 4. As turnaround time of conventional culture is long; therefore, in this study, rapid shell vial culture using Caco-2 cells were evaluated. Moreover, the application of Caco-2 shell vial culture on recovering human metapneumovirus (hMPV) was also investigated.
Materials and methods: This study consisted of four stages. First, recovery of viruses by conventional culture and shell vial culture of Caco-2 were compared. Specimens were added to conventional culture and shell vial simultaneously. For conventional culture, formation of CPE was examined daily and IF staining was performed when CPE was indicated; meanwhile, shell vial culture were incubated for seven days and stained with IF to detect infected cells. In stage two, the effect of incubating shell vial culture in rolling drum was investigated. Shell vials inoculated with the same specimen in duplicate were incubated in rolling drum and without rolling drum simultaneously. IF staining was performed in day 2, and results were obtained. For those which are IF negative in day 2, second shell vial was further incubated to seven days before harvest. In the next stage, a large batch of samples was used to evaluate on the use of Caco-2 shell vial culture in day 2 and day 7. Lastly, Caco-2 shell vial and conventional culture and LLC-MK2 conventional culture were tested for isolation of hMPV.
Results: Compared to Caco-2 conventional culture, recovery rate of shell vial culture was elevated slightly. When experimenting on the effect of incubation in rolling drum, results showed that recovery rate was raised in shell vial with rolling drum in day 2, moreover, the percentage of positive cells were increased significantly (p value < 0.05). Furthermore, in the evaluation of Caco-2 shell vial in day 2 and day 7, 75% of samples were isolated in day 2 while 85% were recovered in day 7. Lastly, in the investigation on recovery of hMPV, 53%, 42% and 17% hMPV positive cases were isolated by Caco-2 shell vial, Caco-2 conventional culture and LLC-MK2 conventional culture respectively.
Conclusion: First, although recovery rate by shell vial and conventional culture were similar, turnaround time was reduced from a week to a few days by shell vial culture. Therefore, Caco-2 shell vial culture is a more efficient than Caco-2 conventional culture in isolating respiratory viruses. The study also showed that incubation of shell vial in rolling drum able to increase the number of positive cells. Furthermore, in this study, Caco-2 cells were also shown to be more efficient in isolating hMPV when compare to LLC-MK2 cells.published_or_final_version
Microbiology
Master
Master of Medical Sciences
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Craig, C. L., Phillip R. Scheuerman, G. R. Lanza, and J. L. Farris. "The Effects of Water Quality Changes Due to Highway Construction on Aquatic Insects as Measured by the DHA-INT Assay." Digital Commons @ East Tennessee State University, 1993. https://dc.etsu.edu/etsu-works/2897.
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Bhatia, Radhika. "Characterisation of the combined effects of physicochemical parameters and toxicants on microbial cells." Thesis, University of Bedfordshire, 2005. http://hdl.handle.net/10547/622046.
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This thesis reports on the combined effects of toxicants and physicochemical factors on micro-organisms. The main objective of the project was to use multi-sensing systems such as mediated and non-mediated sensor systems, growth tests and physicochemical sensors to investigate novel stressor-toxicant-assay combinations. Screen-printed, disposable, developmental-phase, physicochemical sensor constructs (conductivity and dissolved oxygen) were validated under conditions compatible with microbial bioassays, to ascertain their potential role in toxicity testing. The conductivity sensor construct could be used to indirectly inform on the osmolality of the test samples, but the dissolved oxygen sensor construct was not found to give reproducible results. The results were thought to be compromised by in-house screen-printing using a complex carbon ink formulation for the working electrode. Escherichia coli and a consortium with ammonia oxidation capacity (CAOC) were used as the test species for the bioassays. The combined effects of four inorganic salts (NaCl, NaN03, KCl and KN03) and two toxicants (3,5-DCP and HgCh) on E. coli were investigated using the CellSense™ biosensor system, Clark oxygen electrode and microtitre plate growth assays. A variety of trends were observed with each salt-toxicantbioassay combination, emphasising a need for better understanding of the assay media and factors such as bioavailability, to interpret the toxicity data. The results also suggested the importance of using multiple bioassays with varied end points, for toxicity testing. The CAOC, which was isolated from the activated sludge, was tested for physicochemical stressor and toxicant effects using the mediated biosensors. The results were very different from those obtained with E. coli, indicating that each species reacts to toxicants and changes in physicochemical factors differently. Although the full potential of disposable, physicochemical sensors, at the point of toxicity testing was not achieved, the study did investigate previously uncharacterised, combined effects of salts and toxicants on microbial cells. It highlighted the need for development of hybrid systems and also offered a route towards integration of physicochemical and biological sensing systems for simultaneous monitoring of both environmental and biological elements.
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Jacob, Bryant Stephen. "Dynamic DNA Origami Response to SAM Through a Novel Approach with SMK Riboswitches." The Ohio State University, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=osu159826125649515.
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Ashraf, Shamaila. "Studies on infectious bursal disease virus." Connect to resource, 2005. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1124124381.
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Thesis (Ph. D.)--Ohio State University, 2005.Title from first page of PDF file. Document formatted into pages; contains xvi, 216 p.; also includes graphics (some col.). Includes bibliographical references (p. 180-216). Available online via OhioLINK's ETD Center
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Blake, Lynn Dong. "Antimalarial Exoerythrocytic Stage Drug Discovery and Resistance Studies." Scholar Commons, 2016. http://scholarcommons.usf.edu/etd/6182.
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Malaria is a devastating global health issue that affects approximately 200 million people yearly and over half a million deaths are caused by this parasitic protozoan disease. Most commercially available drugs only target the blood stage form of the parasite, but the only way to ensure proper elimination is to treat the exoerythrocytic stages of the parasite development cycle. There is a demand for the discovery of new liver stage antimalarial compounds as there are only two current FDA approved drugs for the treatment of liver stage parasites, one of which fails to eliminate dormant forms and the other inducing hemolytic anemia in patients with G6PD deficiency. In efforts to address the dire need for liver stage drugs, we developed a high-throughput liver stage drug-screening assay to identify liver stage active compounds from a wide variety of chemical libraries with known blood stage activity. The liver stage screen led us to further investigate an old, abandoned compound known as menoctone. Menoctone was developed as a liver stage active antimalarial, however, the development of more potent compounds led to the abandonment of further menoctone research. Our research demonstrated that resistant parasites can transmit mutations through mosquitoes, which was previously believed to not be possible. Furthermore, we studied a novel genetic marker that may indicate potential resistance against malaria parasite infection and the cytotoxic effects associated with the disease. Future experiments aim to identify and advance our methods for the elimination of Plasmodium exoerythrocytic parasites.
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Kawasaki, Yukie. "Aminoglycosides and Syringomycin E as Fungicides Against Fusarium graminearum in Head Blight Disease." DigitalCommons@USU, 2008. https://digitalcommons.usu.edu/etd/202.
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Fusarium graminearum is one of the most problematic phytopathogens in US agriculture. This fungus causes head blight, foot rot, and damping off on wheat and barley. The infection lowers the grain yield and causes contamination of the grain product with mycotoxins. Effective control measures are lacking, and new fungicides that kill F. graminearum but remain safe and economical to use are needed. Newly synthesized aminoglycosides (JL22, JL38, JL39, JL40, NEOF004, NEOF005), classic aminoglycosides (amikacin, gentamicin, kanamycin A, kanamycin B, neomycin, and ribostamycin), and a lipopeptide, syringomycin E (SRE), were studied to determine their antifungal potential to control F. graminearum. Aminoglycosides are protein synthesis inhibitors that mainly target bacteria, but a few were recently observed to kill fungi. They consist of an aminocyclitol ring bound with two or more amino sugars. Novel aminoglycosides were recently synthesized using novel glycodiversification synthetic schemes involving the replacement of the original amino sugars with unusual amino sugars. SRE is an antifungal lipodepsinonapeptide produced by Pseudomonas syringae pv. syringae. This bacterium is an opportunistic pathogen in a wide range of plant species and produces several fungicidal lipopeptides. SRE forms pores on fungal plasma membrane and causes ion fluxes. An enhancement of its antifungal activity is reported in the presence of rhamnolipid surfactants. The antifungal activities of various aminoglycosides, SRE, and a SRE-rhamnolipids mixture were determined against F. graminearum by measuring in vitro minimum inhibition concentrations (MICs) and in planta lesion area and chlorosis development using a leaf infection assay protocol. It was determined that using Tween® 20 at 0.2 % (v/v) concentration in the leaf infection assay promotes lesion development by F. graminearum with minimum phytotoxicity. In vitro, SRE, SYRA, and synthetic aminoglycoside JL38 showed the best antifungal activities. With the in planta assay, all three antifungal agents prevented infection by F. graminearum. However, inconsistent phytotoxicities were observed with SRE and SYRA that were influenced by the Tween® 20 surfactant included in the leaf infection assay. How Tween® 20 induces these phytotoxic inconsistencies is not known.
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bhardwaj, vinay. "Label-free surface-enhanced Raman spectroscopy-linked immunosensor assay (SLISA) for environmental surveillance." FIU Digital Commons, 2015. http://digitalcommons.fiu.edu/etd/2321.
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The contamination of the environment, accidental or intentional, in particular with chemical toxins such as industrial chemicals and chemical warfare agents has increased public fear. There is a critical requirement for the continuous detection of toxins present at very low levels in the environment. Indeed, some ultra-sensitive analytical techniques already exist, for example chromatography and mass spectroscopy, which are approved by the US Environmental Protection Agency for the detection of toxins. However, these techniques are limited to the detection of known toxins. Cellular expression of genomic and proteomic biomarkers in response to toxins allows monitoring of known as well as unknown toxins using Polymerase Chain Reaction and Enzyme Linked Immunosensor Assays. However, these molecular assays allow only the endpoint (extracellular) detection and use labels such as fluorometric, colorimetric and radioactive, which increase chances of uncertainty in detection. Additionally, they are time, labor and cost intensive. These technical limitations are unfavorable towards the development of a biosensor technology for continuous detection of toxins. Federal agencies including the Departments of Homeland Security, Agriculture, Defense and others have urged the development of a detect-to-protect class of advanced biosensors, which enable environmental surveillance of toxins in resource-limited settings.
In this study a Surface-Enhanced Raman Spectroscopy (SERS) immunosensor, aka a SERS-linked immunosensor assay (SLISA), has been developed. Colloidal silver nanoparticles (Ag NPs) were used to design a flexible SERS immunosensor. The SLISA proof-of-concept biosensor was validated by the measurement of a dose dependent expression of RAD54 and HSP70 proteins in response to H2O2 and UV. A prototype microchip, best suited for SERS acquisition, was fabricated using an on-chip SLISA to detect RAD54 expression in response to H2O2. A dose-response relationship between H2O2 and RAD54 is established and correlated with EPA databases, which are established for human health risk assessment in the events of chemical exposure. SLISA outperformed ELISA by allowing RISE (rapid, inexpensive, simple and effective) detection of proteins within 2 hours and 3 steps. It did not require any label and provided qualitative information on antigen-antibody binding. SLISA can easily be translated to a portable assay using a handheld Raman spectrometer and it can be used in resource-limited settings. Additionally, this is the first report to deliver Ag NPs using TATHA2, a fusogenic peptide with cell permeability and endosomal rupture release properties, for rapid and high levels of Ag NPs uptake into yeast without significant toxicity, prerequisites for the development of the first intracellular SERS immunosensor.
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Rojas, Maria J. "Study of Plants Used Against Infections by California Native American Tribes." DigitalCommons@CalPoly, 2020. https://digitalcommons.calpoly.edu/theses/2248.
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The objectives of this research were to evaluate the antibacterial activity and to determine the chemical composition of a list of medicinal plants used by Native Americans in California. Artemisia californica, Mimulus aurantiacus, Equisetum telmateia, Equisetum hyemale, and Marah fabacea were selected from a list of plants reported as having been used for ailments related to infections by tribes located in California. The extracts obtained through steam distillation from E. telmateia, E. hyemale and M. fabacea were assayed for in vitro antibacterial activity against 16 Gram-negative and 6 Gram-positive bacteria using disk diffusion assays and measuring the diameters of inhibition zones. E. telmateia showed the most promising antibacterial activity. The extracts from A. californica, M. aurantiacus and E. telmateia were analyzed for chemical composition, finding eucalyptol, thujone, eugenol, caryophyllene, germacrene D, and propanal as some of the secondary metabolites identified using GC-MS. Our results suggest that E. telmateia can be a potential source for novel antimicrobials against pathogenic bacteria.
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Lowe, Chinn-woan. "A Quadruplex Real-Time PCR Assay for the Rapid Detection and Differentiation of the Burkholderia pseudomallei Complex: B. mallei, B. pseudomallei, and B. thailandensis." BYU ScholarsArchive, 2013. https://scholarsarchive.byu.edu/etd/6247.
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Methods for the rapid detection and differentiation of the Burkholderia pseudomallei complex comprising B. pseudomallei, B. mallei, and B. thailandensis, have been the topic of recent research due to the high degree of phenotypic and genotypic similarities of these species. B. pseudomallei and B. mallei are the causative agents of melioidosis and glanders, respectively. B. pseudomallei and B. mallei are recognized by the CDC as tier 1 select agents. Although B. thailandensis is generally avirulent in mammals, this species displays very similar phenotypic characteristics to that of B. pseudomallei. Optimal identification of these species remains problematic, due to the difficulty in developing a sensitive, selective, and accurate assay. To date, no real-time, multiplex PCR assay has been developed that can detect and differentiate between B. pseudomallei, B. mallei, and B. thailandensis in a single tube format. Here, we describe the development of such an assay that detects and differentiates the species of the B. pseudomallei complex. A real-time quadruplex qPCR assay, Bcom, was designed to target unique genomic regions of B. pseudomallei, B. mallei, B. thailandensis, and the B. pseudomallei complex that detects and differentiates the three species. A total of 299 isolates within the B. pseudomallei complex was evaluated in this study, as well as 15 near-neighbors and other bacterial species. The results showed that this quadruplex assay was capable of detecting the respective species in a given sample at a sensitivity between 288 fg and 277 pg of genomic DNA. The B. pseudomallei- and B. pseudomallei complex-specific assays tested negative on two presumed B. pseudomallei isolates. In addition, a third presumed B. pseudomallei isolate tested negative by the B. pseudomallei-specific test, but was detected by the B. thailandensis and B. pseudomallei complex-specific assays. After cultural and biochemical characterization, 16s rRNA sequencing, and multiple loci sequencing, it is proposed that B. pseudomallei 34 is B. thailandensis 82172 (Accession No. DQ388536), B. pseudomallei Darwin 175 is Elizabethkingia meningoseptica, and B. pseudomallei 135 is a new strain of B. ubonensis 135.
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Thorsell, Mikaela. "Evaluation of C. diff Quik Chek Complete® and comparison with GeneXpert to establish a new diagnostic algorithm." Thesis, Uppsala universitet, Institutionen för kvinnors och barns hälsa, 2018. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-390609.
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Clostridium difficile is the most common antibiotic related diarrhéa disease in Sweden. New recommendations from the Swedish public health authority and European Society of Clinical Microbiology and Infectious Diseases (ESCMID) had led to that a more advanced diagnostic algorithm is of priority. Hence this study, whose purpose was to investigate whether the performance of the rapid test C. diff Quik Chek Complete® could enable the introduction of a new diagnostic algorithm for detection of toxin-forming C. difficile in laboratory medicine in Sundsvall, according to these new recommendations. In the study 119 patient stool-samples were analysed with both GeneXpert and C. diff Quik Chek Complete® and these two combined fulfils these new recommendations of detecting toxin A and B from toxigenic C. difficile together with the enzyme Glutamate Dehydrogenase (GDH) which is produced by all C. difficile stems. The results shows that C. diff Quik Chek Complete® is well matched with GeneXpert and that most of the samples would come to be answered immediately after analysis with C. diff Quik Chek Complete®. The laboratory will save both time and money to establish C. diff Quik Chek Complete® in their algorithm for diagnosing C. difficile infection.
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Hastings, Cody M. "Determination of the effects that a previously uncharacterized secreted product from Klebsiella pneumoniae has on Citrobacter freundii and Enterobacter cloacae biofilms." Digital Commons @ East Tennessee State University, 2017. https://dc.etsu.edu/honors/419.
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More so than ever, Multiple Drug Resistant (MDR) bacteria are on the rise due to overuse of antibiotics along with natural selection for adaptations that enhance drug-resistant properties. One particular bacterial family, Enterobacteriaceae, has been problematic, exhibiting several bacterial members that have developed a precipitous resistance to modern antibiotics and are also primary causative agents of nosocomial, or hospital acquired, infections. Citrobacter freundii (CF) and Enterobacter cloacae (ECL) are two species of the Enterobacteriaceae family causing significant medical concern due to their role in producing numerous opportunistic infections such as bacteremia, lower respiratory tract infections, urinary tract infections, and endocarditis. Adding to the difficulty of this situation is the ability of bacteria to produce biofilms. These biofilms are communities of bacteria that exhibit increased resistance to antibiotic treatment and eradication. Previous work in the laboratory of Dr. Fox at ETSU has identified an uncharacterized product secreted by Klebsiella pneumoniae (KP), another member of the Enterobacteriaceae family, which appears to have inhibitory effects toward CF and ECL. The current study was designed to characterize the effects this secreted product has on CF and ECL biofilms. Through a high throughput microtiter plate assay, the effects of this secreted product were examined on CF and ECL phases of biofilm attachment and maturation. Based on our findings, we have concluded that this secreted product can be categorized as a possible bacteriostatic agent against biofilm cell density, biofilm mass, and cell viability for both biofilm phases of attachment and maturation.
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Merighi, Massimo. "Molecular biology and biochemistry of regulation of Hrp/type III secretion genes in the corn pathogen Pantoea stewartii pv. stewartii." Connect to this title online, 2004. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1069854564.
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Thesis (Ph. D.)--Ohio State University, 2004.Document formatted into pages; contains xxxiii, 421 p. Includes bibliographical references. Abstract available online via OhioLINK's ETD Center; full text release delayed at author's request until 2005 Dec. 2.
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Mody, Shreena Himanshu. "The Antimicrobial Properties of Honey and Their Effect on Pathogenic Bacteria." BYU ScholarsArchive, 2018. https://scholarsarchive.byu.edu/etd/7042.
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Honey has been used to heal wounds since ancient times. There are many references in ancient literature that cite honey for its medicinal uses. It is used as an alternative agent to cure infections of wounds, burns, ulcers etc. Researchers have shown some of the antimicrobial properties of honey when used as an ointment. When applied to an affected area, it helps to promote the growth of healthy tissue. One of the factors on which the quality of the honey depends, is its geographical origin. Based on the location, honey types can vary as much as 100-fold from each other in color, aroma, viscosity, and antimicrobial properties. The important components in honey that play an essential part in healing wounds and contributing to the antimicrobial properties are enzymes. Their presence allows honey to kill various types of pathogenic bacteria, viruses, fungi etc. A higher antimicrobial effect is seen in monofloral honey (when a single plant species is the source of nectar), which is often more potent than other types of honey in terms of antibacterial activity. Resistance of pathogens to these antimicrobial actions has never been shown, which makes honey a more promising source of antimicrobial research. Presently, infections of burns and wounds are very challenging to treat, especially when they are caused by antibiotic-resistant bacteria. The purpose of this study was to examine the antimicrobial properties of honey from Utah and other locales, and to identify promising antimicrobial activities that could be useful in treating infections caused by resistant bacteria.
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de, Oliveira Felipe Marques Souza. "Development and Application of Proximity Assays for Proteome Analysis in Medicine." Doctoral thesis, Uppsala universitet, Molekylära verktyg, 2018. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-334536.
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Along with proteins, a myriad of different molecular biomarkers, such as post-translational modifications and autoantibodies, could be used in an attempt to improve disease detection and progression. In this thesis, I build on several iterations of the proximity ligation assay to develop and apply new adaptable methods to facilitate detection of proteins, autoantibodies and post-translational modifications. In paper I, we present an adaptation of the solid-phase proximity ligation assay (SP-PLA) for the detection of post-translational modification of proteins (PTMs). The assay was adapted for the detection of two of the most commons PTMs present in proteins, glycosylation and phosphorylation, offering the encouraging prospect of using detection of PTMs in a diagnostic or prognostic capacity. In paper II, we developed a variant of the proximity ligation assay using micro titer plate for detection and quantification of protein using optical density as readout in the fluorometer, termed PLARCA. With a detection limit considerably lower than ELISA, PLARCA detected femtomolar levels of these proteins in patient samples. In paper III, we aim to compare detection values of samples collected from earlobe capillary, venous plasma, as well as capillary plasma stored in dried plasma spots (DPS) assessed with a 92-plex inflammation panel using multiplex proximity extension assay (PEA). Despite the high variability in protein measurements between the three sample sources, we were able to conclude that earlobe capillary sampling is a suitable less invasive alternative, to venipuncture. In paper IV, we describe the application of PLARCA and proximity extension assay (PEA) for the detection of GAD65 autoantibodies (GADA). Thus, offering highly sensitive and specific autoimmunity detection.
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Lewis, Ricky W. "TOXICITY OF ENGINEERED NANOMATERIALS TO PLANT GROWTH PROMOTING RHIZOBACTERIA." UKnowledge, 2016. http://uknowledge.uky.edu/pss_etds/77.
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Engineered nanomaterials (ENMs) have become ubiquitous in consumer products and industrial applications, and consequently the environment. Much of the environmentally released ENMs are expected to enter terrestrial ecosystems via land application of nano-enriched biosolids to agricultural fields. Among the organisms most likely to encounter nano-enriched biosolids are the key soil bacteria known as plant growth promoting rhizobacteria (PGPR). I reviewed what is known concerning the toxicological effects of ENMs to PGPR and observed the need for high-throughput methods to evaluate lethal and sublethal toxic responses of aerobic microbes. I addressed this issue by developing high-throughput microplate assays which allowed me to normalize oxygen consumption responses to viable cell estimates. Oxygen consumption is a crucial step in cellular respiration which may be examined relatively easily along with viability and may provide insight into the metabolic/physiological response of bacteria to toxic substances.
Because many of the most toxic nanomaterials (i.e. metal containing materials) exhibit some level of ionic dissolution, I first developed my methods by examining metal ion responses in the PGPR, Bacillus amyloliquefaciens GB03. I found this bacterium exhibits differential oxygen consumption responses to Ag+, Zn2+, and Ni2+. Exposure to Ag+ elicited pronounced increases in O2 consumption, particularly when few viable cells were observed. Also, while Ni2+ and Zn2+ are generally thought to induce similar toxic responses, I found O2 consumption per viable cell was much more variable during Ni2+ exposure and that Zn2+ induced increased O2 utilization to a lesser extent than Ag+. Additionally, I showed my method is useful for probing toxicity of traditional antibiotics by observing large increases in O2 utilization in response to streptomycin, which was used as a positive control due to its known effects on bacterial respiration.
After showing the utility of my method for examining metal ion responses in a single species of PGPR, I investigated the toxicity of silver ENMs (AgENMs) and ions to three PGPR, B. amyloliquefaciens GB03, Sinorhizobium meliloti 2011, and Pseudomonas putida UW4. The ENM exposures consisted of untransformed, polyvinylpyrrolidone coated silver ENMs (PVP-AgENMs) and 100% sulfidized silver ENMs (sAgENMs), which are representative of environmentally transformed AgENMs. I observed species specific O2 consumption responses to silver ions and PVP-AgENMs. Specifically, P. putida exhibited increased O2 consumption across the observed range of viable cells, while B. amyloliquefaciens exhibited responses similar to those found in my first study. Additionally, S. meliloti exhibited more complex responses to Ag+ and PVP-AgENMs, with decreased O2 consumption when cell viability was ~50-75% of no metal controls and increased O2 consumption when cell viability was <50%. I also found the abiotically dissolved fraction of the PVP-AgENMs was likely responsible for most of the toxic response, while abiotic dissolution did not explain the toxicity of sAgENMs.
My work has yielded a straightforward, cost-effective, and high-throughput method of evaluating viability and oxygen consumption in aerobic bacteria. I have used this method to test a broad range of toxic substances, including, metal ions, antibiotics, and untransformed and transformed ENMs. I observed species specific toxic responses to Ag+, PVP-AgENMs, and sAgENMs in PGPR. These results not only show the clear utility of the methodology, but also that it will be crucial to continue examining the responses of specific bacterial strains even as nanotoxicology, as a field, must move toward more complex and environmentally relevant systems.
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Winner, Katherine M. "A fluorescence-based approach to elucidate the subunit arrangement of the essential tRNA deaminase from Trypanosoma brucei." Wittenberg University Honors Theses / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=wuhonors1617803573189193.
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Sun, Eileen. "Investigating Host Protein Function and Developing Assays for Influenza Virus Infection." Thesis, Harvard University, 2015. http://nrs.harvard.edu/urn-3:HUL.InstRepos:17463968.
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With eight genomic segments encoding at least thirteen proteins, influenza virus can subvert a cell into a virus-producing factory. To study influenza infection, I utilize versatile fluorescent-based technologies to non-invasively probe a range of biological processes and targets in both static and living systems. This thesis covers three broad areas aimed towards unraveling the complexities of influenza infection: understanding how host proteins regulate viral infection, developing assays to study infection heterogeneity, and applying live cell imaging to study antiviral mechanism of action.
First, I discuss how two cellular proteins—COPI complex and CD81—facilitate influenza infection. Genome-wide knockdown screens identified COPI complex and CD81 as influenza host dependency factors, but their specific function remained unclear. Applying imaging and flow cytometry methods, I found COPI siRNA knockdown inhibited virus entry during internalization and transport to late endosomes, and late stage infection during viral membrane protein trafficking. However, acute pharmacological treatment only recapitulated membrane protein trafficking defects, suggesting COPI directly facilitates late stage infection, not entry. In contrast, CD81 facilitates both viral fusion during entry and scission during egress. Single particle tracking studies revealed ~50% of virus particle fusion events occurred within CD81+ endosomes, and CD81 is recruited to progeny virus budding zones to facilitate viral scission.
Second, while influenza infection encompasses a complex mixture of incomplete infection events, no influenza infection assay has thus far comprehensively evaluated infection heterogeneity. To study the prevalence and composition of incomplete infection events, I developed a multiplexed immunofluorescence assay to probe viral protein expression from all eight genomic segments. I found that influenza infection heterogeneity is highly prevalent, and the composition of different infection states exhibits correlated expression patterns amongst a subset of viral proteins.
Lastly, I include a study applying live cell imaging to dissect the mechanism of a newly identified influenza antiviral drug, clotrimazole. I found clotrimazole inhibited WSN influenza infection between viral fusion and replication. Altogether, a multi-pronged approach is required to study the complex influenza infection cycle. Deciphering the complexities will guide development of much needed influenza therapeutics and vaccines.Medical Sciences
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Lam, Jonathan Lam. "Identification of mammalian cell signaling in response to plasma membrane perforation: Endocytosis of Listeria monocytogenes and The Repair Machinery." The Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1543497502225763.
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Graffagna, Barry. "Virus Production and Cell Viability of HSV-1-infected Murine Keratinocytes (HEL-30) Co-cultured with Murine Macrophages (RAW 264.7)." Wright State University / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=wright1542212790178886.
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Coyle, Peter Valentine. "Development and application of serodiagnostic assays for the investigation of human herpesvirus 6 infection." Thesis, Queen's University Belfast, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.333774.
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Hamed, Aljali. "Development of IgE microarray assays for classification of allergic individuals." Thesis, University of Nottingham, 2016. http://eprints.nottingham.ac.uk/37431/.
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Allergic reactions are pathological immune reactions against common, harmless environmental proteins, termed “allergens”. Type I hypersensitivity reactions are mediated by IgE antibodies; these reactions are the most prevalent, affecting all ages and both genders to almost the same extent. This form of allergic reaction is distributed widely affecting more than 25% of the population in the industrial world. The increased incidence of IgE-mediated allergy is thought to be a combination of environmental and genetic factors, each account for about 50%. The effects associated with this disease may reduce quality of life, daily activities, and in some cases threaten life, hence affecting work and leisure time, increasing healthcare and medication costs. IgE-mediated allergy is defined a complex innate and adoptive immune response to natural harmless environmental allergens in which allergen- specific IgE antibodies are predominantly produced. In addition, it is believed that other immunoglobulin isotypes might be involved in atopic diseases (mainly IgG4), although the roles of these antibody isotype are still debatable whether they are pathogenic or protective antibodies. Generally, diagnostic procedures for IgE-mediated allergy begin with the patient’s history, clinical symptoms, physical examination, in vivo provocation tests, and are eventually confirmed with laboratory investigations. In this thesis we develop a microarray technology for use in-house for comprehensive investigations of immunoglobulin reactivity profiles against numerous allergens in atopic disease. Several printing and blocking buffers were developed in-house to preserve functionality of the arrayed proteins, minimise background noise, and increase signal intensity. The new diagnostic platform was validated by set of quality controls, then applied on large-scale applications (allergic patients, general populations, suspected asthmatics and healthy control individuals) to measure total and specific IgE antibodies. Application of the developed antibody microarray platform identified the difficulty of setting cutoffs using a “healthy control group”. We tested both the 75th and the 95th percentiles of the healthy control group as a cut-off point for total IgE levels that resulted in 39.3ng/ml and 70.68ng/ml respectively. When the 75th percentile was selected, the number of participants in the allergy patients classed as positive above the cutoff points was 92 out of 105 (87.6%). However, if the 95th percentile was selected, the number of positive patients was 63 out of 105 (60%) in the same group. On the other hand, the developed allergen microarray platform was used to immunoprofile allergen specific immunoglobulin subtypes in the four different cohorts against 70 SPT allergens. MeV software was used to present the reactivity profiles of the immunoglobulin subtypes semi-quantitatively that resulted in the hierarchical clustering according to allergen species. The reactivity profiles of allergen specific IgE in the allergic group recognized multiple allergens while specific IgE in the healthy control group was negative except for one serum sample. Specific IgE in the random population was lower than in the suspected asthmatics although it recognized allergens from different species (ingested, inhaled, venom). Similarly, this multi-allergen microarray platform was adapted to investigate the roles of other antibody isotypes (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2) in IgE- mediated allergy. The study revealed no roles for these isotype except IgG4 that showed multiple recognition patterns in serum samples of healthy individuals and allergic patients. A comparison between microarray results and provocation testing (SPT) was performed for the samples from suspected asthmatics. To enable direct comparison, IgE:IgG4 ratio was utilized as IgE levels alone would not be expected to correlate with wheal and flare reaction. The IgE:IgG4 ratio showed good correlation with the six allergens tested by SPT (r=0.9299, p<0.0001). In addition, the multi-allergen chip can detect additional responses outside the standard six SPT- allergens performed routinely, and moreover reports on IgG4 responses that are never assessed by SPT, but are potentially important to the likelihood of asthma and allergy. Overall, the results indicated that the new developed microarray platform is highly specific, sensitive and reproducible. Subsequently, this high-throughput microarray platform can work as a diagnostic tool for monitoring the development of allergic diseases, predicting allergic reactions particularly for polysensitised patients, evaluating patient response to treatments, and eventually allowing the classification of allergic individuals. In conclusion, a new high-throughput multi-allergen platform has been developed that will certainly improve the future of allergy diagnosis.
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Murray, Aimee Kaye. "Selection for antibiotic resistance in the aquatic environment : novel assays to detect effect concentrations of micropollutants." Thesis, University of Exeter, 2017. http://hdl.handle.net/10871/30325.
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The environment is increasingly recognised as a key player in the emergence and mobilisation of antibiotic resistance, which negatively impacts human health, healthcare systems, and farming practices worldwide. Recent work has demonstrated concentrations of antibiotics in the natural environment may select for resistance in situ, but a scarcity of meaningful data has prevented rigorous environmental risk assessment of antibiotics. Without such data, mitigation strategies, such as improved antibiotic stewardship or environmental discharge limits, cannot be effectively designed or implemented. This thesis designed and developed two methods for determining effect concentrations of antibiotics in complex microbial communities, thereby generating a significant amount of data to address this knowledge gap. Minimal selective concentrations (MSCs) were determined in long term selection experiments for four classes of antibiotic at concentrations as low as 0.4 μg/L, which is below many measured environmental concentrations. Lowest observed effect concentrations were determined using a short term, growth based assay which were highly predictive of MSCs. A novel finding was significant selection for cefotaxime resistance occurred at a wide range of antibiotic concentrations, from 125 μg/L - 64 mg/L, which has important clinical implications. Determination of MSC in single species assays was also shown to be a poor predictor of MSC in a complex microbial community. Co-selection for antimicrobial resistance was demonstrated in selection experiments and through improved understanding of class 1 integron evolution, assessing selective effects on resistance gene acquisition using a novel PCR method and next-generation sequencing. In the final study, a novel resistance determinant (UDP-galactose 4-epimerase) conferring cross-resistance to biocides and antibiotics was discovered, providing a target for further study. These findings indicate selection and co-selection for antimicrobial resistance is likely to occur in the environment, and provides the means to rapidly generate further data to aid in the development of appropriate mitigation strategies.
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Morgan, M. K., Phillip R. Scheuerman, C. S. Bishop, and Rebecca A. Pyles. "Teratogenic Potential of Atrazine and 2,4-D Using Fetax." Digital Commons @ East Tennessee State University, 1996. https://dc.etsu.edu/etsu-works/2871.
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The teratogenic potential of commercial formulations of atrazine (40.8%) and 2,4-D was evaluated using FETAX (frog embryo teratogenic assay--Xenopus). Because these herbicides have been detected in ground and surface water, this study was designed to determine the adverse effects in buffer and natural water for both herbicides. All treatments showed a significant concentration-response effect on exposed embryos, except for the 2,4-D natural water sample. Atrazine (solubility of the commercial formula used 70 mg/L at 20 degrees C), compared to 2,4-D (solubility = 311 mg/L at pH = 1 and 25 degrees C), had a significantly greater teratogenic effect in both the buffer (atrazine EC50 = 33 mg/L, LC50 = 100 mg/L, TI = 3.03; 2,4-D EC50 = 245 mg/L, LC50 = 254 mg/L, TI = 1.04) and natural water samples (atrazine EC50 < 8 mg/L, LC50 = 126 mg/L; 2,4-D EC50 and LC50 > 270 mg/L). The 2,4-D EC50 and LC50 values for the buffer were similar at 245 mg/L and 254 mg/L. These similar values and the teratogenic index (TI) of 1.04 suggested that 2,4-D was more embryotoxic than teratogenic to frog embryos at high concentrations. Atrazine in natural water demonstrated a significantly greater EC50 (100% abnormality at 8 mg/L, the lowest test concentration) to frog embryos than the buffer experiment (EC50 = 33 mg/L). The extrapolated lowest observable adverse effect concentration (LOAEC) for the natural water experiment was 1.1 mg/L. These results suggest that atrazine toxicity is enhanced by the synergistic or additive effects of some component of the water or atrazine was already present in the sample. In contrast to atrazine, 2,4-D was less toxic in natural water than buffer. These results suggest that both atrazine and 2,4-D pose little threat, since their embryotoxicity and teratogenicity to frog embryos occur at high concentrations approaching their maximum solubility levels in water.
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Case, J., Phillip R. Scheuerman, C. Bishop, and A. Hougland. "An Evaluation of Microbial Enzyme Assays as an Indicator of Pollution in Stream Sediments." Digital Commons @ East Tennessee State University, 1997. https://dc.etsu.edu/etsu-works/2913.
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Du, Preez Martella. "Development of novel seminested polymerase chain reaction assays for detecting toxigenic Vibrio cholerae and Shigella spp. in water." Diss., University of Pretoria, 2001. http://hdl.handle.net/2263/26893.
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Jumani, Rajiv Satish. "Methods To Identify And Develop Drugs For Cryptosporidiosis." ScholarWorks @ UVM, 2018. https://scholarworks.uvm.edu/graddis/892.
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Cryptosporidiosis is a common diarrheal disease caused by intestinal infection with the apicomplexan parasite Cryptosporidium, in humans usually either with C. hominis or C. parvum. Unfortunately, given a large burden of disease in children and immunocompromised people like AIDS patients, the only currently approved treatment, nitazoxanide, is unreliable for these patient populations. To address the urgent need for new drugs for the most vulnerable populations, large phenotypic screening efforts have been established to identify anti-Cryptosporidium growth inhibitors in vitro (hits). However, in the absence of a gold standard drug, the in vitro and in vivo characteristics that should be used to prioritize screening hits are not known. This thesis is focused on identifying promising anti-Cryptosporidium hits and drug leads, and using them to establish validated methods to guide hit-to-lead studies for anti-Cryptosporidium drug development.
A re-analysis of our phenotypic screen of the Medicines for Malaria Venture Open Access Malaria Box identified a promising C. parvum growth inhibitor, MMV665917. It had similar in vitro activity against C. hominis, C. parvum Iowa, and C. parvum field strains, and it was amenable to preliminary structural activity relationship studies using commercially available variants, with one variant demonstrating nanomolar potency. Furthermore, MMV665917 was effective in vivo in an acute interferon-γ mouse model of cryptosporidiosis; and it appeared to cure an established infection in the chronic NOD SCID gamma (NSG) mouse model, unlike nitazoxanide, paromomycin, and clofazimine. We hypothesized that anti-Cryptosporidium activity in the highly immunocompromised chronic NSG mouse model might relate to compounds being capable of killing and eliminating parasites (cidal), rather than only preventing growth (static). To test this, we developed a novel in vitro parasite persistence assay that showed that MMV665917 was potentially cidal, whereas nitazoxanide, paromomycin and clofazimine appeared static. This pharmacodynamic assay also provided the concentration of compound required to maximize rate of parasite elimination, which could help design in vivo experiments.
To further characterize compounds based on mechanism of action, we developed a range of in vitro medium-throughput life-stage assays. To validate and gain value from the assays, a “learner set” of compounds from our in-house screens and collaborations were tested in all of the in vitro assays and in the in vivo NSG mouse model. Using these assays, it was possible to group molecules based on chemical class/mechanism of action. Because compounds from distinct groups showed activity in the NSG mouse model, these methods could be used to obtain a diverse set of early-stage Cryptosporidium inhibitors for prioritization. Furthermore, compounds that appeared static in the in vitro parasite persistence assay did not have activity in the NSG mouse model. In summary, we report the identification and development of a highly promising initial lead, MMV665917, and report a range of in vitro assays that can be used to prioritize anti-Cryptosporidium hits and leads.