This thesis documents Phase III of a research program undertaken by the Virginia Transportation Research Council and the Virginia Tech Transportation Institute in cooperation with the 3M Company and the Virginia Department of Transportation for the evaluation of visual performance of retroreflective signs of various color combinations. Phase I was an off-road field experiment conducted to determine the best sign color combination, letter stroke width, and letter size for the emergency sign. Based upon the results of Phase I, three color combinations were chosen for testing (black on coral, black on light blue, and yellow on purple) against a baseline color combination of black on orange.Phase II was conducted using an instrumented vehicle through a construction zone-related detour. Questionnaire data were also obtained. The independent variables of interest were sign color combination, age, and visibility condition. The findings of Phase II indicated that use of a color combination other than the traditional black on orange sign would improve driver performance and safety when used for trailblazing during critical incidents, especially when the incident route overlaps a work zone detour. A serious limitation of Phases I and II is that the use of fluorescent colors was not evaluated. Anecdotal evidence suggests that the use of fluorescent colors on signs improves their conspicuity. The purpose of Phase III was to evaluate fluorescent sign color combinations for incident management trailblazing purposes. This study consisted of an on-road investigation using an instrumented vehicle over a 12.2-mile route in urban and rural areas of Montgomery County, Virginia. The following conclusions were made:*A non-fluorescent yellow on non-fluorescent purple sign is least preferred by both older and younger drivers when compared to the other sign color combinations employed in this study.*Both younger and older drivers have a preference for a black on fluorescent yellow-green sign.* Fewer late braking maneuvers and fewer turn errors were recorded during daytime conditions than during nighttime conditions. *Older drivers tended to register more late braking maneuvers than did younger drivers. / Master of Science

La première partie de ces travaux de thèse concerne la synthèse d’acides aminés phosphole par formation d’une liaison P-C. Les propriétés de fluorescence (absorption, émission et rendement quantique) sont modulées à la fois par les différents substituants présents sur le phosphore (BH3, O, S…) ainsi que par le squelette aromatique du phosphole. Des couplages peptidiques modèles réalisés en solution et sur support solide démontrent la possibilité d’intégrer ces acides aminés dans des peptides d’intérêts. La deuxième partie concerne la synthèse de nouveaux phospholes fonctionnalisés ainsi que d’une nouvelle méthode d’accrochage pour les introduire sur différents groupes pendants (SH, NH2, OH) d’acide aminés et peptides via la formation de liaisons P-S, P-N ou P-O. La troisième partie de ce travail a consisté en la synthèse d’une nouvelle classe d’acides aminés tétraphénylsilole fluorescents qui présentent des propriétés d’AIE (aggregation-induced emission) et pourront être utilisés pour le marquage de peptides d’intérêts. / The first part of this work is focused on phospholyl amino acids synthesis by formation of a P-C bond. The fluorescent properties (absorption, emission and quantum yield) are modulated either by the substituent on the phosphorus atom (BH3, O, S, …) or by the aromatic skeleton of the phosphole. Peptide coupling in solution or on solid support were performed and showed the possibility to introduce such amino acids into peptide of interest. The second part of this work is dedicated to the synthesis of new functionalized phospholes for a chemoselective grafting on amino acid and peptides pendant groups (SH, NH2, OH) via PS, P-N or P-O bonds. The third part consists into the synthesis of a new class of tetraphenylsilole amino acids which exhibit AIE (aggregation-induced emission) fluorescent properties. Those compounds were successfully incorporated into di- an tri- peptides in solution and on solid support.

The use of fluorescent imaging probes for the real time detection of cellular malfunctions, such as enzyme over expression has shown promise. Fluorescent dyes with absorption and emission values below 600 nm are limited in their in vivo applications due to high background auto-fluorescence and low resolution images. Employing near infrared (NIR) fluorophores such as cyanine dyes can overcome this disadvantage. Cyanine dyes can be synthesised using solution or solid-phase techniques with the use of solution phase chemistry allowing for larger scale and higher yielding reactions. Utilising a selection of functional groups and varying polymethine chain lengths a cyanine dye library with tuneable absorption and emission wavelengths was synthesised. This thesis gives the first detailed examples of how modifications on heptamethine cyanine dyes alter their cellular uptake and cellular toxicity. Furthermore, a NIR fluorescent microsphere is reported as well as NIR functionalised microspheres with the ability to be tracked within cells. Additional lines of work involved the synthesis of a fluorescent sensor for the visualisation of bacteria. Aminopeptidases are present within the peptidoglycan cell wall of Gram negative bacteria and therefore can be targeted for real time detection of bacteria to aid in the detection of infectious diseases. A coumarin based probe is reported which detects aminopeptidase in gram negative bacteria in vitro.

In the current work, the photophysics and photochemistry of the phototoxic red fluorescent protein (RFP) KillerRed was investigated. KillerRed's phototoxicity makes it useful for studying oxidative stress on cell physiology and for cell killing in photodynamic therapy. Spectroscopic probes were used to show that the phototoxicity of KillerRed stems primarily from a type I photosensitization mechanism producing radicals. The production of radicals was supported by electron paramagnetic resonance (EPR) studies, where a long-lived radical was observed in KillerRed and two other RFPs (mRFP and DsRed) following excitation. Transient absorption spectroscopy, various other spectroscopic techniques, and the published crystal structure of KillerRed indicate that the long-filled water channel is likely responsible for the increased phototoxicity of KillerRed. In the blue fluorescent protein (BFP) mKalama1, some of the same techniques were applied to understand the photophysics and photochemistry on the timescale ranging from femtoseconds to seconds. Transient absorption spectroscopy and previously published results demonstrate that two-photon excitation of mKalama1 likely results in the formation of a radical cation and solvated electrons. This may explain the blinking behavior which has been observed on the single molecule level for many fluorescent proteins, the identity of which has remained elusive. It was also shown that the chromophore, while neutral in the ground state, does not exhibit excited-state proton transfer (ESPT) during its nanosecond excited-state lifetime; however, the chromophore undergoes a deprotonation in the ground state after electronic relaxation. This work plays a key role in our understanding of fluorescent proteins and will help pave the way to developing new ones. The research on the BFPs was extended to improve them for cellular imaging. This was accomplished by identification of dark states in the BFPs which are longer in wavelength than the collected fluorescence. Using dual lasers, it was shown that these dark states could be optically depleted, thereby increasing the overall fluorescence without enhancing the background fluorescence. Rational site-directed mutagenesis was carried out on the BFPs and the mutants were screened for fluorescence enhancement. These proteins were then analyzed using transient absorption spectroscopy to elucidate the identity of the dark state(s) used for fluorescence enhancement.

Coatings are often used as a means of protecting aluminum alloy and steel structures in industry. The assessment of corrosion under these coatings can be challenging. Corrosion sensing coatings can exhibit properties that allow undercoating corrosion to be identified before it can be seen with the naked eye. This would be very advantageous and could potentially result in tremendous savings in time and money when structures undergo routine maintenance. Our work involved the study of corrosion sensing coatings with incorporated fluorescent indicators that can be used to sense the undercoating corrosion on metal substrates. The fluorescent indicator in the coated-aluminum system was a negative indicator, i.e. the indicator in the coating was initially fluorescent and subsequently non-fluorescent due to the reduced pH at the anodic sites of corrosion. The fluorescent indicator in coated-steel system was positive, in the sense that the coating changed from non-fluorescent to fluorescent over the cathodic areas due to increased pH. The corrosion sensing coating was composed of commercial epoxy-polyamide and the indicator: 7-amino-4-methylcoumarin (7-AMC) for the coated-aluminum alloy system and 7-diethylamino-4-methylcoumarin (7-DMC) for the coated-steel system. The feasibility of using 7-AMC for sensing early undercoating corrosion was demonstrated by using fluorescent observations, Electrochemical Impedance Spectroscopy (EIS), Scanning Electron Microscope (SEM) and Energy Dispersive Spectroscopy (EDS) tests. EIS results estimated that with continuous immersion the undercoating corrosion occurred within 24 hours after immersion in the salt solution. When corrosion occurred, the corrosion was invisible under natural light. However, small spots appeared in the fluorescent image, changing from initially fluorescent to non-fluorescent where the anodic sites were identified by SEM and EDS. In other words, the fluorescent indicator could sense the early undercoating corrosion, although blistering can be a competing mechanism associated with corrosion under some conditions. The sensitivity of the 7-AMC corrosion detection system was tested by applying anodic current to the metal and measuring the charge at which fluorescence quenching was detected. The critical charge for a detectable pit under the coating was approximately 2x10⁻⁵ C, which implied a critical radius of a single corrosion spot or set of spots of approximately 10 [mu]m. The fluorescent properties of 7-AMC, its effect on the protectiveness, its sensitivity to pH and its concentration in the coating are explored as well. Fourier transform spectroscopy (FTIR) was used to characterize the structure of the coating with and without 7-AMC. The results suggested that there is no structure change occurring after adding 7-AMC into the coating. Fluorescence behavior, electrochemical behavior, microscopic evidence, and visual observations of coated steel specimens with 7-DMC are compared based on exposure to saltwater conditions. Some of the challenges associated with the use of these types of coatings will be presented. This includes the interference from the increased production of ferrous and ferric ions. All of this information is aimed at the development of corrosion sensing coatings that can reveal undercoating corrosion before it is visible to the naked eye. / text

The molecular cloning and subsequent engineering of the green fluorescent protein (GFP) of the jellyfish Aequoria victoria allowed a novel approach to the investigation of cell signalling. GFP and its mutants can now not only be used to target specific organelles in living cells but also function as a basis for a variety of sensors for biologically important ions and molecular interactions. GFP-based Ca2+- sensors have been successfully used for studies in mammalian and plant cells. In filamentous fungi, however, they have not yet been reported to work. Since only little is known about calcium signalling in filamentous fungi, this project aimed to improve existing GFP-based Ca2+- sensors by exchanging the original fluorophores for improved versions and expressing those in the filamentous fungus Aspergillus niger. During this project, the donor and acceptor fluorophores of 3 existing Ca2+-FRETprobes based on cameleons and troponin C-sensors, have been changed, 2 novel positive FRET controls have been designed and these , as well as donor and acceptor fluorophores alone, have been expressed in the filamentous fungus Aspergillus niger. The probes were assessed using different imaging techniques, such as conventional confocal laser scanning microscopy (CLSM), fluorescence lifetime imaging microscopy (FLIM) and spectral imaging using a Leica TSC SP5 confocal and IRIS, a novel spectral imaging device designed at Heriot Watt University. Problems were encountered that prevented FRET analysis using CLSM and IRIS. These were due mainly to the difference in expression level of the constructs and the distribution of the emission bandpasses of the IRIS system. Analysis of the spectral data obtained on the Leica confocal system and analysis of the FLIM results, however, revealed significant differences between the donor only and the positive FRET controls. Spectra of the positive FRET controls and the Ca2+-sensitive probes showed emission peaks of both the donor and the acceptor fluorophores upon excitation of the donor fluorophore alone while analysis of the FLIM results revealed an additional decay component in the positive FRET controls. Both results are very strong indicators that we can detect FRET in living hyphae of Aspergillus niger transformed with the probes designed during this project.

Photoreactive groups have been used in photoaffinity labelling of chemical macromolecules via the generation of highly reactive species upon short wave light irradiation. One of the most efficient photoreactive functional groups is trifluoromethyl aryldiazirine (TFMAD). This compound was synthesised as part of the work discussed in this thesis, making use of microwave irradiation to shorten reaction times (Chapter I). An investigation of properties allowed the development of three different applications for conjugation to biomolecules. The first application consisted of the development of an approach for generation of small-molecule microarrays, where a 2,000 compound library was immobilised onto the glass surface through carbene insertion. The microarray was then used to screen for potential binders to beta-transducin repeat containing protein (b-TrCP1) allowing the reduction of possible candidates to less than 25 compounds (Chapter II). The second application was the synthesis of two probes to allow the selective delivery of active compounds inside specific organelles or cells. The diazirine moiety was used as a rapid way to covalently capture a number of cargos. The approach allowed a peptoid and an anticancer drug to be conjugated to the two probes and their cell penetrability properties and therapeutic effect were studied, respectively (Chapter III). Finally, the insertion properties of TFMAD were used to develop approaches to attach DNA onto microspheres and the efficiency of this delivery system was evaluated (Chapter IV).

Narrowly dispersed amino-functionalised polystyrene microspheres, with a range of diameters, were successfully synthesised via emulsion and dispersion polymerisation. Fluorescent labelling allowed cellular translocation to be assessed in a variety of cell lines and was found to be very high, but controllable, whilst exhibiting no detrimental effect on cellular viability. In order to fully determine the mode of microsphere uptake, “beadfected” melanoma (B16F10) cells were studied using both chemical and microscopic methods. Uptake was found to be wholly unreliant upon energetic processes, with microspheres located cytoplasmically and not encapsulated within endosomes, an important characteristic for delivery devices. In order to demonstrate the effective delivery of exogenous cargo mediated by microspheres, short interfering (si)-RNAs were conjugated to beads and investigated for the gene silencing of enhanced green fluorescent protein (EGFP) in cervical cancer (HeLa) and embryonic (E14) stem cells. EGFP knockdown was found to be highly efficient after 48 – 72 hours. Dual-functionalised microspheres displaying a fluorophore (Cy5) and siRNA allowed only those cells beadfected with the delivery vehicle (and thus containing siRNA) to be assessed for EGFP expression, yielding an accurate assessment of microsphere-mediated gene silencing. In addition, by manipulation of the microsphere preparation conditions, micro-doughnuts and paramagnetic microspheres were produced and their cellular uptake assessed. Paramagnetic microspheres were found to enter cells efficiently and were subsequently used to bias the movement of beadfected cells in response to an externally applied magnet, while micro-doughnuts were found to exhibit cell selective properties and were noted to traffic specifically to the liver in vivo.

This particular work is proposed as a test of the expedience of using the fluorescent-antibody technique as a method for enumeration and identification of certain strains of B. cereus that have been found to be effective in preventing taste and odor in water supplies resulting from certain Actinomycete blooms.

Thesis advisor: Larry W. McLaughlin / Nucleic acids are fundamental biomolecules responsible for all activities of a living cell. DNA serves as an instruction manual to the cell, containing blueprints and directions for all cellular processes, while RNA serves to carry out the messages held within DNA. Research into the structure, stability, and function of nucleic acids has revealed much about the origin and evolution of life. The ultimate goal of this work is to understand how molecules bind and associate within the minor groove of double stranded, helical DNA. A series of 2'-deoxyadenosine analogues are modified at the three position by replacing the N3-nitrogen with carbon. Substitution at this position is designed to emulate the effects of removing hydrogen bond acceptors, introducing steric bulk, and tethering functional groups of interest into the minor groove. These functional groups mimic small molecules that have been shown to bind within the minor groove of A-T rich sequences as well as serve as a platform for further substitution by fluorescent tags. The synthetic effort needed to obtain purine nucleosides containing each of these modifications was non-trivial. New methodologies unveiled directing and protecting strategies towards the desired isomer of these modified nucleosides in higher yields than those previously deemed acceptable. Application of these modified nucleosides into duplex DNA reveals thermodynamic parameters for how small molecules bind to the minor groove and the effects of introducing biomarkers into an unprecedented region of DNA. / Thesis (PhD) — Boston College, 2011. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.