Thesis (M.S.)--Ohio University, August, 1980. / Title from PDF t.p.

Thesis (Ph. D.)--University of Texas at Austin, 2002. / Vita. Includes bibliographical references. Available also from UMI Company.

Not available / text

The development of new drug therapies traditionally requires mass screening of thousands if not millions of substances to identify lead compounds. They are then further optimised to increase potency. The screening of the large pharmaceutical compound libraries can be incredibly expensive, with the industry responding by miniaturising the assays to smaller formats, enabling the compound screening to be automated and, importantly, eliminating assay reagents that are a major contributing cost for running large screens. A potential target for such an approach is the genetic recoding site of viruses like HIV-1 and SARS. They use programmed recoding of the genetic code to regulate the translation of necessary proteins required for viable virus production. For example HIV-1 uses a -1 frameshift mechanism to regulate the ratio of the Gag to the Pol proteins, crucial for viable virus formation. The study of recoding, including readthrough of premature termination codons have most recently used bicistronic reporters with different combinations of enzymes. The most widely used plasmid bicistronic reporter utilises a dual luciferase arrangement comprised of firefly luciferase and Renilla luciferase reporters flanking the DNA being studied. Both of the luciferase enzymatic reporters emit light in response to their respective substrates. The cost of these substrates is the major issue to using luciferase reporters for high throughput screening. My study aimed at designing and developing a bicistronic assay suitable for genetic recoding that was amenable to high throughput screening. The luciferase reporters were replaced with Green Fluorescent Protein (GFP) reporters that do not require the addition of substrates. The development of a dual GFP assay required the appropriate selection of GFP fluorophores, the best arrangement of the GFPs to maximise the ratio of relative fluorescence intensity signal to background, the optimisation of the cells and growth conditions, DNA transfection, plate reader selection, and optical filter sets. Cassettes encoding protein linkers were also incorporated into the design of the constructs to separate the fluorescent proteins spatially to facilitate unimpaired folding into their functional units within the fusion protein. The assay was further improved by moving from transient transfection to stably expressing cell lines. A viable assay was almost achieved for 96 (and 384) well plates with a Z� factor compatible with the assay being suitable for high throughput screening. The assay was used to test a small collection of compounds known to interact with the ribosome and compounds known in the literature to affect frameshifting. This proof of concept was important, since it showed that the assay, with the various modifications, optimisations and miniaturisation steps, still retained the capability of correctly measuring the -1 frameshifting efficiency at the HIV-1 recoding site, and recording compound-induced modulations to the frameshifting efficiency. The compounds cycloheximide and anisomycin, for example, were shown to decrease -1 frameshifting albeit at some expense to overall protein synthesis. The dual GFP assay was also shown to be able to measure accurately changes in the frameshift efficiency brought about by mutations to the frameshift element, and additionally, it would be suitable for the detection and study of compounds, like the recently reported PTC-124 (currently undergoing phase II clinical trial for Duchenne Muscular Dystrophy and cystic fibrosis) that increases readthrough of a UGA premature stop codon mutation. The dual GFP assay developed in this study is at most only 1/10th of the cost of a comparable dual luciferase assay, largely due to removal of assay substrates and transfection reagents. The assay has a robust Z� factor comparable to that of the dual luciferase assay, and would substantially decrease the costs of high throughput screening in situations where a bicistronic reporter is required. The HIV-1 frameshift element is such a site.

The development of new drug therapies traditionally requires mass screening of thousands if not millions of substances to identify lead compounds. They are then further optimised to increase potency. The screening of the large pharmaceutical compound libraries can be incredibly expensive, with the industry responding by miniaturising the assays to smaller formats, enabling the compound screening to be automated and, importantly, eliminating assay reagents that are a major contributing cost for running large screens. A potential target for such an approach is the genetic recoding site of viruses like HIV-1 and SARS. They use programmed recoding of the genetic code to regulate the translation of necessary proteins required for viable virus production. For example HIV-1 uses a -1 frameshift mechanism to regulate the ratio of the Gag to the Pol proteins, crucial for viable virus formation. The study of recoding, including readthrough of premature termination codons have most recently used bicistronic reporters with different combinations of enzymes. The most widely used plasmid bicistronic reporter utilises a dual luciferase arrangement comprised of firefly luciferase and Renilla luciferase reporters flanking the DNA being studied. Both of the luciferase enzymatic reporters emit light in response to their respective substrates. The cost of these substrates is the major issue to using luciferase reporters for high throughput screening. My study aimed at designing and developing a bicistronic assay suitable for genetic recoding that was amenable to high throughput screening. The luciferase reporters were replaced with Green Fluorescent Protein (GFP) reporters that do not require the addition of substrates. The development of a dual GFP assay required the appropriate selection of GFP fluorophores, the best arrangement of the GFPs to maximise the ratio of relative fluorescence intensity signal to background, the optimisation of the cells and growth conditions, DNA transfection, plate reader selection, and optical filter sets. Cassettes encoding protein linkers were also incorporated into the design of the constructs to separate the fluorescent proteins spatially to facilitate unimpaired folding into their functional units within the fusion protein. The assay was further improved by moving from transient transfection to stably expressing cell lines. A viable assay was almost achieved for 96 (and 384) well plates with a Z� factor compatible with the assay being suitable for high throughput screening. The assay was used to test a small collection of compounds known to interact with the ribosome and compounds known in the literature to affect frameshifting. This proof of concept was important, since it showed that the assay, with the various modifications, optimisations and miniaturisation steps, still retained the capability of correctly measuring the -1 frameshifting efficiency at the HIV-1 recoding site, and recording compound-induced modulations to the frameshifting efficiency. The compounds cycloheximide and anisomycin, for example, were shown to decrease -1 frameshifting albeit at some expense to overall protein synthesis. The dual GFP assay was also shown to be able to measure accurately changes in the frameshift efficiency brought about by mutations to the frameshift element, and additionally, it would be suitable for the detection and study of compounds, like the recently reported PTC-124 (currently undergoing phase II clinical trial for Duchenne Muscular Dystrophy and cystic fibrosis) that increases readthrough of a UGA premature stop codon mutation. The dual GFP assay developed in this study is at most only 1/10th of the cost of a comparable dual luciferase assay, largely due to removal of assay substrates and transfection reagents. The assay has a robust Z� factor comparable to that of the dual luciferase assay, and would substantially decrease the costs of high throughput screening in situations where a bicistronic reporter is required. The HIV-1 frameshift element is such a site.

Thesis (M.S. in Operations Research)--Naval Postgraduate School, March 1980. / Thesis Advisor(s): Neil, Douglas. Second Reader: Moroney, William. "March 1980." Description based on title screen as viewed on May 25, 2010. DTIC Descriptor(s): Data Displays, Man Machine Systems, Cockpits, Performance (Human), Reaction Time, Pilots, Analysis Of Variance, Theses, Coding, Colors, Errors, Symbols, Cathode Ray Tube Screens, Color Vision, Alphanumeric Displays DTIC Identifier(s): Color Coding. Author(s) subject terms: Coding Techniques, Symbols,Colors, Reaction Time, Performance, Errors, Alphanumerics. Includes bibliographical references (p. 65-66). Also available in print.

Indirect detection digital imaging systems used in medical imaging, compromises powder phosphor scintillators as X-ray to light converters. Powder phosphors should combine image quality and light output parameters in order to produce high quality diagnostic images, with the parallel dose reduction to the patient. Additionally, they must be characterized by short decay times in order to be used in digital breast tomosynthesis (DBT) and dual energy imaging (DE). The aim of the present PhD thesis is the investigation of the optimum powder phosphor scintillator for use in a CMOS based digital imaging system and the investigation of the combination of the digital imaging system with the optimum scintillator for low energy medical applications, like DBT. Scintillating screens, were prepared using the method of sedimentation, by Lu2SiO5:Ce, Gd2O2S:Eu and Gd2O2S:Tb powder phosphors. Their properties were evaluated by experimentally determining parameters related to optical signal intensity and distribution at the scintillator exit surface, characterizing medical image quality and the patient’s dose. By comparing the luminescence efficiency and image quality properties of Lu2SiO5:Ce, Gd2O2S:Eu and Gd2O2S:Tb, the scintillating screen with the optimum characteristics was defined and placed, in close contact with the CMOS photodiode. MTF and DQE of our CMOS sensor were found better at the whole spatial frequency range with previously published data for a passive CMOS sensor, while NNPS was comparable. The evaluated CMOS sensor is characterized by high spatial resolution and detection efficiency properties that make it suitable for DBT. Additionally, image quality is acceptable at low exposure levels, which is crucial in DBT and DE applications where high patient’s dose is a drawback for the establishment of these methods. / Τα ψηφιακά συστήματα ιατρικής απεικόνισης, έμμεσης ανίχνευσης, χρησιμοποιούν φωσφόρους σπινθηριστές ως μετατροπείς της ακτινοβολίας-Χ σε ορατό φως. Οι φώσφοροι σπινθηριστές πρέπει να συνδυάζουν χαρακτηριστικά ποιότητας εικόνας και απόδοσης σε φωταύγεια προκειμένου να παράγουν εικόνες υψηλής διαγνωστικής αξίας με τη παράλληλη ελάττωση της δόσης στον εξεταζόμενο. Επιπλέον πρέπει να έχουν μικρούς χρόνους απόσβεσης για χρήση σε συστήματα ψηφιακής τομοσύνθεσης (DBT) και απεικόνισης διπλής ενέργειας (DE). Σκοπός της παρούσας Διδακτορικής Διατριβής ήταν η διερεύνηση των βέλτιστων υλικών φωσφόρων σπινθηριστών για χρήση σε ολοκληρωμένο ψηφιακό απεικονιστικό σύστημα τύπου CMOS καθώς και η διερεύνηση των χαρακτηριστικών του συστήματος ψηφιακού ανιχνευτή/ βέλτιστου σπινθηριστή για ιατρικές εφαρμογές χαμηλών ενεργειών, όπως η DBT. Παρασκευάστηκαν, με τη μέθοδο της καθίζησης, φθορίζουσες οθόνες από υλικά σπινθηριστών όπως τα Lu2SiO5:Ce, Gd2O2S:Eu και Gd2O2S:Tb. Οι ιδιότητες τους μελετήθηκαν αξιολογώντας πειραματικά παραμέτρους οι οποίες εκφράζουν την ένταση και την κατανομή του παραγόμενου σήματος στην έξοδο του ανιχνευτή και σχετίζονται άμεσα με την ποιότητα της ιατρικής εικόνας αλλά και με τη δόση στον εξεταζόμενο. Στον ανιχνευτή τοποθετήθηκε, σε άμεση επαφή με τις φωτοδιόδους CMOS, φθορίζουσα οθόνη Gd2O2S:Tb, η οποία προσδιορίστηκε μέσω της σύγκρισης των ανωτέρω υλικών σε απόδοση φωταύγειας και ποιότητα εικόνας. Η απόδοση του CMOS ήταν καλύτερη εν συγκρίσει με δημοσιευμένα αποτελέσματα για αισθητήρα PPS CMOS, σε όλο το εύρος των χωρικών συχνοτήτων. Τα αποτελέσματα αυτά δείχνουν ότι ο υπό εξέταση ανιχνευτής τύπου CMOS έχει υψηλή διακριτική ικανότητα και ανιχνευτική αποδοτικότητα, κρατώντας παράλληλα χαμηλά επίπεδα θορύβου καθιστώντας τoν κατάλληλο για χρήση σε πρότυπο σύστημα (DBT). Επιπλέον βρέθηκε ότι η ποιότητα εικόνας δεν υποβαθμίζεται σε χαμηλά επίπεδα έκθεσης, στοιχείο που είναι σημαντικό για εφαρμογές (DBT) και (DE), όπου η αυξημένη δόση στον εξεταζόμενο είναι ανασταλτικός παράγοντας για τη καθιέρωση και ευρεία αποδοχή των συγκεκριμένων μεθόδων.

Fluorescent x-ray screens are used in medical x-ray diagnostics to absorb x-ray photons and convert these x-ray photons to visible light. The light distribution from these screens are then registered on photographic film to give an x-ray image. Both the sensitivity and the resolution characteristics of these systems are dependent on the x-ray photon energy. To enable a study of these and some other energy dependent characteristics of x-ray screens a number of almost monoener-getic radiation sources were constructed, tested with regard to their purity and calibrated. Both film and a photo-multiplier tube were used as light detectors.The sensitivity of screens with three different screen phosphors were studied as a function of the photon energy and large variations in sensitivity was found for different photon energies and screen phosphors. The light from the screens has been compared to the absorbed energy in the screens and this comparison shows that the energy dependence of the screens can approximately be predicted by calculations of the absorbed energy, except at low photon energies where other effects like increased light absorption in the screens is present.The modulation transfer factor (MTF) was studied both experimentally and theoretically as a function of photon energy. Two effects were shown to influence the energy dependence of the MTF. At low energies an increased light diffusion will destroy the MTF and at energies above the K-edge of the high-Z elements in the screens the production and re-absorption of K-radiation will deteriorate the MTF.Both the energy dependence of the screen-film sensitivity and the MTF have been calculated for some normally used spectral distributions from x-ray tubes and significant changes due to choice of kV and filtration of the beam were found. Other effects such as the number of interacting photons in the screens per unit area, contribution of K-radiation from one screen to the other, and light contribution to the front emulsion of the film compared to the back emulsion have also been investigated as a function of photon energy.Optimization of x-ray systems and clinical routines to give the lowest possible radiation dose to the patient with an acceptable image quality is an important task to carry out. The energy dependent characteristi es of x-ray screens studied in this work is a lead in the optimizing of the system with regard to choice of x-ray screens, film and radiation quality. / digitalisering@umu