Dissertations / Theses on the topic 'Fluorescent screens'

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.

Listeria monocytogenes is a Gram-positive, facultative intracellular pathogen that causes gastroenteritis, which in the young, the elderly, and the immunocompromised can progress to severe invasive disease with high mortality. While previous studies have largely elucidated the bacterial and host mechanisms necessary for the bacterium to access its replicative niche in the host cell cytosol, the L. monocytogenes factors required for adaptation to life within this restrictive environment are poorly understood. In this dissertation, I describe a fluorescence-activated cell sorting (FACS)-based differential fluorescence genetic screening technique for the identification of L. monocytogenes genes necessary for optimal intracellular replication. Bacteria harboring deletions in identified genes were defective for intracellular replication, plaque formation, and in vivo virulence, validating the ability of the screening method to identify novel intracellular replication-defective mutants. Minor alteration of the FACS-based screening strategy allowed the detection and differentiation of bacterial mutants displaying varying severities of actin-based motility defects. A preliminary FACS-based genetic screen to identify actin-based motility mutants isolated multiple independent insertions within internal control genes, demonstrating the potential utility of FACS-based differential fluorescence genetic screening methods for the identification of L. monocytogenes genes important for multiple virulence phenotypes. Lastly, my characterization of the X-prolyl aminopeptidase PepP, a novel virulence factor identified by the FACS-based genetic screen to discover genes necessary for optimal intracellular replication, revealed this enzyme plays an unexpected role in L. monocytogenes virulence gene regulation.

Alternative splicing of the vascular endothelial growth factor A (VEGF-A) terminal exon generates two protein families with differing functions. Pro-angiogenic VEGFxxx isoforms are produced via selection of the proximal 3’ splice site of the terminal exon. Use of an alternative distal splice site creates the anti-angiogenic VEGFxxxb proteins. A bichromatic splicing-sensitive reporter was designed to mimic VEGF alternative splicing and used as a molecular tool to further investigate this alternative splicing event. The terminal exon and preceding intron of VEGF were inserted into a minigene construct followed by the coding sequences for two fluorescent proteins. A different fluorescent protein is expressed depending on which 3’ splice site of the exon is used during splicing. The fluorescent output can be used to follow splicing decisions in vitro and in vivo. Following successful reporter validation in different cell lines and altering splicing using known modulators, small pilot screens were undertaken to search for novel regulators of the splicing decision that creates pro-/anti-angiogenic VEGF isoforms. A larger screen was performed using a library of 1280 small molecules (LOPAC), all compounds of the library were pharmacologically active and have known biological targets. Alterations to reporter splicing were measured using a fluorescent plate reader to detect RFP and GFP expression. Compounds of interest were further validated using flow cytometry and assessed for effect on endogenous VEGF alternative splicing. In vitro angiogenesis assays were used to demonstrate anti-angiogenic effect. Anti-angiogenic activity and the effect on tumour growth were investigated in several in vivo models.

Two main subjects, ellipsometry and computer screen photo-assisted techniques (CSPT), form the main line in this thesis. Ellipsometry is an optical technique based on the detection of polarization changes of light upon interaction with a sample. As most optical detection techniques it is non-intrusive and an additional advantage is its high surface sensitivity: thickness resolution in the order of pm can in principle be achieved. Therefore, ellipsometry is widely used as a technique for determination of optical constants and layer thickness for thin-layer structures. Lately ellipsometry has also been proposed for sensing applications, utilizing the detection of changes in the properties of thin layers. One application is described in this thesis concerning the detection of volatile organic solvents in gas phase using modified porous silicon layers, fabricated by electrochemical etching of silicon to create nm-sized pores. This greatly increases the surface area, promoting gas detection because the number of adsorption sites increases. Other applications of ellipsometry discussed in this thesis are based on combination with CSPT. CSPT is a way to exploit existing optical techniques for use in low-cost applications. In CSPT the computer screen itself is used as a (programmable) light source for optical measurements. For detection a web camera can be used and the whole measurement platform is formed by the computer. Since computers are available almost everywhere, this is a promising way to create optical measurement techniques for widespread use, for example in home-diagnostics. Since the only thing that needs to be added is a sample holder governing the physical or chemical process and directing the light, the cost can be kept very low. First, the use of CSPT for the measurement of fluorescence is described. Fluorescence is used in many detection applications, usually by chemically attaching a fluorescent marker molecule to a suitable species in the process and monitoring the fluorescent emission. The detection of fluorescence is shown to be possible using CSPT, first in a cuvette-based setup, then using a custom designed micro array. In the latter, polarizers were used for contrast enhancement, which in turn led to the implementation of an existing idea to test CSPT for ellipsometry measurements. In a first demonstration, involving thickness measurement of silicon dioxide on silicon, a thickness resolution in the order of nm was already achieved. After improvement of the system, gradients in protein layers could be detected, opening the door toward biosensor applications. Some further development will be needed to make the CSPT applications described here ready for the market, but the results so far are certainly promising.

This diploma thesis deals with automatic detection and measurement of the electron beam in the images from a transmission electron microscope (TEM). The introduction provides a description of the construction and the main parts of the electron microscope. In the theoretical part, there are summarized modes of illumination from the fluorescent screen. Machine learning, specifically convolution neural network U-Net is used for automatic detection of the electron beam in the image. The measurement of the beam is based on ellipse approximation, which defines the size and dimension of the beam. Neural network learning requires an extensive database of images. For this purpose, the own augmentation approach is proposed, which applies a specific combination of geometric transformations for each mode of illumination. In the conclusion of this thesis, the results are evaluated and summarized. This proposed algorithm achieves 0.815 of the DICE coefficient, which describes an overlap between two sets. The thesis was designed in Python programming language.

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.

Yes
α2,8-Linked polysialic acid (polySia) is an oncofoetal antigen with high abundance during embryonic development. It reappears in malignant tumours of neuroendocrine origin. Two polysialyltransferases (polySTs) ST8SiaII and IV are responsible for polySia biosynthesis. During development, both enzymes are essential to control polySia expression. However, in tumours ST8SiaII is the prevalent enzyme. Consequently, ST8SiaII is an attractive target for novel cancer therapeutics. A major challenge is the high structural and functional conservation of ST8SiaII and -IV. An assay system that enables differential testing of ST8SiaII and -IV would be of high value to search for specific inhibitors. Here we exploited the different modes of acceptor recognition and elongation for this purpose. With DMB-DP3 and DMB-DP12 (fluorescently labelled sialic acid oligomers with a degree of polymerisation of 3 and 12, respectively) we identified stark differences between the two enzymes. The new acceptors enabled the simple comparative testing of the polyST initial transfer rate for a series of CMP-activated and N-substituted sialic acid derivatives. Of these derivatives, the non-transferable CMP-Neu5Cyclo was found to be a new, competitive ST8SiaII inhibitor.

The dominant powder scintillator in most medical imaging modalities for decades is Gd2O2S:Tb due to the very good intrinsic properties and overall efficiency. Except for Gd2O2S:Tb there are alternative powder phosphor scintillators like Lu2SiO5:Ce and Gd2O2S:Eu that has been suggested for use in various medical imaging modalities. Gd2O2S:Eu emits red light and can be combined mainly with digital imaging devices like CCDs and CMOS based detectors. The purposes of the present thesis, is to investigate the fundamental imaging performance of a high resolution CMOS based imaging sensor combined with custom made Europium (Eu3+) activated Gd2O2S screens in terms of Modulation Transfer Function (MTF), Normalized Noise Power Spectrum (NNPS), Detective Quantum Efficiency (DQE), Noise Equivalent Quanta (NEQ) and Information Capacity (IC) covering the mammography and general radiography energy ranges. The CMOS sensor was coupled to two Gd2O2S:Eu scintillator screens with coating thicknesses of 33.3 and 65.1 mg/cm2, respectively, which were placed in direct contact with the photodiode array. The CMOS photodiode array, featuring 1200x1600 pixels with a pixel pitch of 22.5 m , was used as an optical photon detector. In addition to frequency dependent parameters (MTF, NPS, DQE) characterizing image quality, image information content was assessed through the application of information capacity (IC). The MTF was measured using the slanted-edge method to avoid aliasing while the Normalized NPS (NNPS) was determined by two-dimensional (2D) Fourier transforming of uniformly exposed images. Both parameters were assessed by irradiation under the RQA-5 protocol (70kVp digital-radiography) recommended by the International Electrotechnical Commission Reports 62220-1 and the W/Rh, W/Ag beam qualities (28kVp digital-mammography). The DQE was assessed from the measured MTF, NNPS and the direct entrance surface air-Kerma (ESAK) obtained from X-ray spectra measurement with a portable cadmium telluride (CdTe) detector. The spectral matching factor between the optical spectra emitted by the Gd2O2S:Eu and the Gd2O2S:Tb screens and the CMOS optical sensor, evaluated in the present study, was 1 and 0.95 respectively. The ESAK values ranged between 11.2-87.5 Gy , for RQA-5, and between 65.8-334 Gy , for W/Rh, W/Ag beam qualities. It was found that the detector response function was linear for the exposure ranges under investigation. Under radiographic conditions the MTF of the present system was found higher than previously published MTF data for a 48 m CMOS sensor, in the low up to medium frequency ranges. DQE was found comparable, while the NNPS appeared to be higher in the frequency range under investigation (0–10 cycles/mm). NEQ reached a maximum (73563 mm-2) in the low frequency range (1.8 cycles/mm), under the RQA 5 (ESAK: 11.2 Gy ) conditions. IC values were found to range between 1730-1851 bits/mm2. Under mammographic conditions MTF, NNPS and NEQ were found comparable to data previously published for the 48 m CMOS sensor while the DQE was found lower. The corresponding IC values were found ranging between 2475 and 2821 bits/mm2. The imaging performance of europium (Eu3+) activated Gd2O2S screens in combination to the CMOS sensor, investigated in the present study, was found comparable to those of Terbium (Tb) activated Gd2O2S screens (combined with the CMOS sensor). It can be thus claimed that red emitting phosphors could be suitably used in digital imaging systems, where the Silicon (Si) based photodetectors are more sensitive to longer wavelength ranges, and particularly in the red wavelength range.
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Cyclic nucleotide-gated channels (CNGCs) are non-selective cation channels that were first identified in vertebrate photosensory and olfactory neurons. Although the physiological roles and biophysical properties of animal CNGCs have been well studied, much less is known about these channels in plants. The Arabidopsis genome encodes twenty putative CNGC subunits that are postulated to form channel complexes that mediate various physiological processes involving abiotic and biotic stress responses, ion homeostasis and development. The identification of Arabidopsis autoimmune CNGC mutants, such as defense no death class (dnd1 and dnd2), and the constitutive expressor of pathogenesis related genes 22 (cpr22) implicate AtCNGC2, 4, 11 and 12 in plant immunity. Here, I present a comprehensive study of the molecular mechanisms involved in CNGC-mediated signaling pathways with emphasis on pathogen defense. Previously, a forward genetics approach aimed to identify suppressor mutants of the rare gain-of-function autoimmune mutant, cpr22, identified key residues that are important for CNGC subunit interactions and channel function. First, I present a structure-function analysis of one of these suppressor mutants (S58) that revealed a key residue in the cyclic nucleotide binding domain involved in the stable regulation of CNGCs. Second, I present a new suppressor screen using AtCNGC2 T-DNA knockout mutants that specifically aimed to identify novel downstream components of CNGC-mediated pathogen defense signaling. In this screen, I successfully isolated and characterized the novel Arabidopsis mutant, repressor of defense no death 1 (rdd1), and expanded this study to demonstrate its involvement in AtCNGC2 and AtCNGC4-mediated signal transduction. Additionally, I demonstrated for the first time, the physical interaction of AtCNGC2 and AtCNGC4 subunits in planta. The findings presented in this thesis broaden our current knowledge of CNGCs in plants, and provide a new foundation for future elucidation of the structure-function relationships and signal transduction mediated by these channels.