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Title: CONFOCAL LASER SCANNING MICROSCOPY AS A TOOL FOR THE INVESTIGATION OF TETRACYCLINE FLUORESCENCE IN ARCHAEOLOGICALHUMAN BONE.
Creator: Maggiano, Corey, Waterman, Jane, University of Central Florida
Abstract / Description: Fluorochromes such as tetracycline have been used to label bone for histomorphometric analysis, measuring bone formation, growth, maintenance, and pathology. More recently, similar fluorescence has been observed in ancient human bone. Attributed to tetracycline (TC) exposure, this phenomenon could affect various aspects of health during life and/or preservation of remains postmortem. Standard epifluorescence microscopy is the most common tool employed in the analysis of these labels. Though...
Show moreFluorochromes such as tetracycline have been used to label bone for histomorphometric analysis, measuring bone formation, growth, maintenance, and pathology. More recently, similar fluorescence has been observed in ancient human bone. Attributed to tetracycline (TC) exposure, this phenomenon could affect various aspects of health during life and/or preservation of remains postmortem. Standard epifluorescence microscopy is the most common tool employed in the analysis of these labels. Though valuable, this technique is limited by its inability to penetrate bone three-dimensionally and its inclusion of out-of-focus light, possibly disrupting accurate analysis. Confocal Laser Scanning Microscopy (CLSM) has been demonstrated as a valuable tool for three-dimensional histology. Its application to the study of compact bone fluorescence has been lacking, especially in archaeological and forensic sciences. In the following two papers, modern TC-controlled bone is compared to well preserved archaeological bone recovered from the Dakhleh Oasis, Egypt, using both standard wide-field and more modern confocal techniques for imaging and analysis. Spectral analysis via CLSM shows that both modern and ancient fluorescent labels in bone share the exact same fluorescence emission peak at 525 nm. Differences in the shape of the spectral curve and photobleaching characteristics are discussed. In addition, CLSM's high-resolution two- and three-dimensional imaging capabilities (in polarized light, scattered light, and fluorescence light) are found to increase the flexibility and creativity of investigations into the occurrence of tetracycline labels in archaeological bone and could have added benefits for modern medical and anatomical experimentation.
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Date Issued: 2005
Identifier: CFE0000836, ucf:46680
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0000836

Title: Fluorescence Off-On Sensors for F-, K+, Fe3+, and Ca2+ Ions.
Creator: Sui, Binglin, Belfield, Kevin, Miles, Delbert, Zou, Shengli, Frazer, Andrew, Bhattacharya, Aniket, University of Central Florida
Abstract / Description: Fluorescence spectroscopy has been considered to be one of the most important research techniques in modern analytical chemistry, biochemistry, and biophysics. At present, fluorescence is a dominant methodology widely used in a great number of research domains, including biotechnology, medical diagnostics, genetic analysis, DNA sequencing, flow cytometry, and forensic analysis, to name just a few. In the past decade, with the rapid development of fluorescence microscopy, there has been a...
Show moreFluorescence spectroscopy has been considered to be one of the most important research techniques in modern analytical chemistry, biochemistry, and biophysics. At present, fluorescence is a dominant methodology widely used in a great number of research domains, including biotechnology, medical diagnostics, genetic analysis, DNA sequencing, flow cytometry, and forensic analysis, to name just a few. In the past decade, with the rapid development of fluorescence microscopy, there has been a considerable growth in applying fluorescence technique to cellular imaging. The distinguished merits of fluorescence techniques, such as high sensitivity, non-invasiveness, low cytotoxicity, low cost, and convenience, make it a promising tool to replace radioactive tracers for most biochemical measurements, avoiding the high expense and difficulties of handling radioactive tracers.Among the wide range of applications of fluorescence technique, fluorescent sensing of various cations and anions is one of the most important and active areas. This dissertation is all about developing fluorescent sensors for physiologically significant ions, including F-, K+, Fe3+, and Ca2+. All of these sensors demonstrate fluorescence (")turn-on(") response upon interacting with their respective ions, which makes them much more appealing than those based on fluorescence quenching mechanisms.In Chapter II, a novel highly selective fluorescence turn-on F- sensor (FS), comprised of a fluorene platform serving as the chromophore, and two 1,2,3-triazolium groups functioning as the signaling moieties, is described. The function of FS is established on the basis of deprotonation of the C-H bonds of 1,2,3-triazolium groups, which makes FS the first reported anion sensor based on the deprotonation of a C-H bond. Easy-to-prepare test strips were prepared for determining F- in aqueous media, providing an inexpensive and convenient approach to estimate whether the concentration of F- contained in drinking water is at a safe level.Chapter III contains an optimized synthesis of a reported K+-selective group (TAC), and the development of two TAC-based fluorescence turn-on K+ sensors (KS1 and KS2). The synthetic route of TAC is shortened and its overall yield is enhanced from 3.6% to 19.5%. Both KS1 and KS2 exhibited excellent selectivity toward K+ over other physiological metal cations, high sensitivity for K+ sensing, and pH insensitivity in the physiological pH range. Confocal fluorescence microscopy experiments demonstrate that they are capable of sensing K+ within living cells. 2PA determination reveals that KS2 has a desirable 2PA cross section of 500 GM at 940 nm, which makes it a two-photon red-emitting fluorescent sensor for K+.Chapter IV describes the development of a novel BODIPY-based fluorescence turn-on Fe3+ sensor (FeS). FeS is a conjugate of two moieties, a BODIPY platform serving as the fluorophore and a 1,10-diaza-18-crown-6 based cryptand acting as the Fe3+ recognition moiety. FeS displays good selectivity, high sensitivity, reversibility, and pH insensitivity toward Fe3+ sensing. Based on its excellent performance in determining Fe3+ and very low cytotoxicity, FeS was effectively applied to sensing Fe3+ in living cells.In Chapter V, a new BODIPY-based fluorescence turn-on sensor (CaS) was designed and synthesized for selectively and sensitively determining Ca2+. CaS is comprised of two moieties, a BODIPY fluorophore and a Ca2+ complexing unit. CaS demonstrated selective fluorescence turn-on response towards Ca2+ over other biological metal cations. Moreover, CaS exhibited desirable sensitivity for Ca2+ detection, which makes it more suitable for extracellular Ca2+ determination. In addition, CaS was insensitive to the pH of the physiological environment, especially in the pH range of blood and serum. Therefore, CaS has potential to be applied to sensing Ca2+ ions in extracellular environments.Chapter VI discusses potential future work of KS2 and CaS, following the results achieved in this dissertation. Based on the desirable performances of both sensors in sensing their respective ions, future work could largely be focused on their applications in cellular imaging.
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Date Issued: 2014
Identifier: CFE0005888, ucf:50883
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0005888

Title: Planar Laser Induced Fluorescence Experiments and Modeling Study of Jets in Crossflow at Various Injection Angles.
Creator: Thompson, Luke, Vasu Sumathi, Subith, Kassab, Alain, Kapat, Jayanta, University of Central Florida
Abstract / Description: Planar Laser Induced Fluorescence (PLIF) with acetone seeding was applied to measure the scalar fields of an axisymmetric freejet and an inclined jet-in-crossflow as applicable to film cooling. From the scalar fields, jet-mixing and trajectory characteristics were obtained. In order to validate the technique, the canonical example of a nonreacting freejet of Reynolds Numbers 900-9000 was investigated. Desired structural characteristics were observed and showed strong agreement with...
Show morePlanar Laser Induced Fluorescence (PLIF) with acetone seeding was applied to measure the scalar fields of an axisymmetric freejet and an inclined jet-in-crossflow as applicable to film cooling. From the scalar fields, jet-mixing and trajectory characteristics were obtained. In order to validate the technique, the canonical example of a nonreacting freejet of Reynolds Numbers 900-9000 was investigated. Desired structural characteristics were observed and showed strong agreement with computational modeling. After validating the technique with the axisymmetric jet, the jet-in-crossflow was tested with various velocity ratios and jet injection angles. Results indicated the degree of wall separation for different injection angles and demonstrate both the time-averaged trajectories as well as select near-wall concentration results for varying jet momentum fluxes. Consistent with literature findings, the orthogonal jet trajectory for varying blowing ratios collapsed when scaled by the jet-to-freestream velocity ratio and hole diameter, rd. Similar collapsing was demonstrated in the case of a non-orthogonal jet. Computational Fluid Dynamic (CFD) simulations using the OpenFOAM software was used to compare predictions with select experimental cases, and yielded reasonable agreement. Insight into the importance and structure of the counter rotating vortex pair and general flow field turbulence was highlighted by cross validation between CFD and experimental results.
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Date Issued: 2015
Identifier: CFE0006057, ucf:50992
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0006057

Title: Multidimensional Room-Temperature Fluorescence Microscopy for the Nondestructive Analysis of Forensic Trace Textile Fibers.
Creator: Mujumdar, Nirvani, Campiglia, Andres, Sigman, Michael, Harper, James, Rex, Matthew, Peale, Robert, University of Central Florida
Abstract / Description: The purpose of this dissertation is to advance nondestructive methodology for forensic fiber examination. Non-destructive techniques that can either discriminate between similar fibers or match a known to a questioned fiber (-) and still preserve the physical integrity of the fibers for further court examination - are highly valuable in forensic science. A challenging aspect of forensic fiber examinations involves the comparison of fibers colored with visually indistinguishable dyestuffs....
Show moreThe purpose of this dissertation is to advance nondestructive methodology for forensic fiber examination. Non-destructive techniques that can either discriminate between similar fibers or match a known to a questioned fiber (-) and still preserve the physical integrity of the fibers for further court examination - are highly valuable in forensic science. A challenging aspect of forensic fiber examinations involves the comparison of fibers colored with visually indistinguishable dyestuffs. This is not an uncommon situation, as there are numerous indistinguishable fibers pre-dyed with commercial dyes of virtually identical colors. Minimal chemical structural variations are actually encouraged by the dye patent process and commercial competition.The common denominator to forensic methodology is the fact that fiber analysis primarily focuses on the dyes used to color the fibers and do not investigate other potential discriminating components present in the fiber. This dissertation explores a different aspect of fiber analysis as it focuses on the total fluorescence emission of fibers. In addition to the contribution of the textile dye (or dyes) to the fluorescence spectrum of the fiber, we consider the contribution of intrinsic fluorescence impurities (-) i.e. impurities imbedded into the fibers during fabrication of garments - as a reproducible source of fiber comparison. Although fluorescence microscopy is used in forensic labs for single fiber examination, measurements are made with the aid of band-pass filters that provide very limited information on the spectral profiles of fibers. We take the non-destructive nature of fluorescence microscopy to a higher level of selectivity with the collection of room-temperature fluorescence excitation emission matrices (RTF-EEMs). The information contained in the EEMs was first used to select the best excitation wavelength for recording first order data, i.e. two-dimensional fluorescence spectra. Pairwise comparisons involved the following visually indistinguishable fibers: nylon 361 pre-dyed with acid yellow (AY) 17 and AY 23, acrylic 864 pre-dyed with basic green (BG) 1 and BG 4, acetate satin 105B pre-dyed with disperse blue (DB) 3 and DB 14, and polyester 777 pre-dyed with disperse red (DR) 1 and DR 19. With the exception of acrylic 864 fibers dyed with BG1 and BG4, the comparison of two-dimensional spectra via principal component analysis (PCA) provided accurate fiber identification for all the analyzed fibers. The same approach was later applied to the investigation of laundering effects on the comparison of textile fibers. The presence of brighteners and other detergent components adsorbed in the fibers provided spectral fingerprints that enhanced the fiber identification process.The full dimensionality of EEMs was then explored with the aid of parallel factor analysis (PARAFAC), a second order algorithm capable to determine the number of fluorescence components that contribute to an EEM along with their individual excitation and emission profiles. The application of PARAFAC was carried out unsupervised and supervised by linear discrimination analysis (LDA). The classification performances of PARAFAC and LDA-supervised PARAFAC were compared to the one obtained with supervised discriminant unfolded partial least squares (DU-PLS). The best discrimination was obtained with the supervised DU-PLS, which allowed the pairwise differentiation of the four pairs of investigated fibers.DU-PLS was then used to investigate weathering effects on the spectral features of cotton 400 pre-dyed with DB1, nylon 361 pre-dyed with AY17 and acrylic 864 pre-dyed with BG4. The investigated fibers were exposed to humid (Florida) and dry (Arizona) weathering conditions for three, six, nine and twelve months. In all cases, this algorithm was unable to differentiate non-exposed acrylic fibers from exposed acrylic fibers. DU-PLS was able to differentiate non-exposed cotton and nylon fibers from exposed fibers to Florida and Arizona weathering conditions. It was possible to determine the period of exposure to either Florida or Arizona conditions. It was also possible to discriminate between fibers exposed to Florida or Arizona weathering conditions for the same period of time. These results provide the foundation for future studies towards a non-destructive approach capable to provide information on the history of the fiber.
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Date Issued: 2016
Identifier: CFE0006838, ucf:51773
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0006838

Title: A NOVEL SETUP FOR HIGH-PRESSURE RAMAN SPECTROSCOPY UNDER A MICROSCOPE.
Creator: Oakeson, Thomas, Schulte, Alfons, University of Central Florida
Abstract / Description: Functional properties of biological molecules and cells are affected by environmental parameters such as temperature and pressure. While Raman spectroscopy provides an intrinsic probe of molecular structural changes, the incorporation of a microscope enables studies of minuscule amounts of biological compounds with spatial resolution on a micron scale. We have developed a novel setup which combines a Raman microscope and a high pressure cell. A micro-capillary made out of fused silica...
Show moreFunctional properties of biological molecules and cells are affected by environmental parameters such as temperature and pressure. While Raman spectroscopy provides an intrinsic probe of molecular structural changes, the incorporation of a microscope enables studies of minuscule amounts of biological compounds with spatial resolution on a micron scale. We have developed a novel setup which combines a Raman microscope and a high pressure cell. A micro-capillary made out of fused silica simultaneously serves as the supporting body and the optical window of the pressure cell. The cell has been tested over the pressure range from 0.1 to 4 kbar. Raman spectra of less than 100 nanoliter amount of amino acid and protein solutions have been measured in the micro-capillary high pressure cell. It is also demonstrated that the setup is well suited for spectrally resolved fluorescence measurements at variable pressure.
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Date Issued: 2007
Identifier: CFE0001683, ucf:47208
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0001683

Title: ROOM TEMPERATURE FLUORESCENCE SPECTROSCOPY AS A TOOL FOR THE FORENSIC TRACE ANALYSIS OF TEXTILE FIBERS.
Creator: Rex, Matthew, Campiglia, Andres, University of Central Florida
Abstract / Description: ABSTRACT Trace textile fiber evidence is found at numerous crime scenes and plays an important role in linking a suspect to the respective scene. Several methods currently exist for the analysis of trace fiber evidence. Microscopy provides information regarding the fibers material, color and weave. For more detailed chemical analysis chromatographic methods are employed and for discrimination between dyes, liquid chromatography coupled with mass spectrometry (LC-MS) is currently the method...
Show moreABSTRACT Trace textile fiber evidence is found at numerous crime scenes and plays an important role in linking a suspect to the respective scene. Several methods currently exist for the analysis of trace fiber evidence. Microscopy provides information regarding the fibers material, color and weave. For more detailed chemical analysis chromatographic methods are employed and for discrimination between dyes, liquid chromatography coupled with mass spectrometry (LC-MS) is currently the method providing the most discrimination. These methods have primarily focused on the dyes used to color the fibers and have not investigated other components that can potentially discriminate among fibers. This dissertation deals with investigations into the fluorescence of the fiber dyes, (contaminants?) and the fibers themselves, as well as methodology for discriminating between fibers using fluorescence. Initial systematic analysis was conducted on dye standards and extracts taken from fibers colored with the respective dyes of interest. Absorbance, excitation and fluorescence spectra were compared between standards and extracts to determine the optimal area of the fiber to investigate: dyes, fluorescent impurities or the whole fiber. High performance liquid chromatography investigations were performed to give detailed information on the number of dye and fluorescent components present in extracts. Our investigations then focused on the best room-temperature fluorescence (RTF) data format for analysis and discrimination of fiber samples. An excitation emission matrix (EEM) was found to give the greatest amount of spectral information and provide the highest level of discrimination. Successful discrimination between non similar and similar fibers was achieved with the aid of Chemometric analysis. The level of discrimination obtained via RTF-EEM spectroscopy was sufficient to differentiate among fibers obtained from two separate cloths of the same material and colored with the same dye reagent. Final studies deal with examining exposure of the fiber to various environmental contaminants. Clothing fibers are typically exposed to myriad numbers of contaminants, from food stains to cigarette smoke. The challenge then becomes detecting fluorescence signals from trace amounts of these environmental contaminants. We demonstrate the detection and classification of polycyclic aromatic hyrdrocarbons (PAH) present on fibers after exposure to cigarette smoke. This dissertation also investigates the change in fluorescence emission after laundering fibers numerous times. The main drawback of chemical analysis of fibers is the destructive nature of the methods. To extract a dye or contaminant from a fiber essentially destroys the evidence. This leaves the investigator without their original sample in the courtroom. This also provides a finite amount of sample for testing and analysis. This is true of chromatographic methods and for the method detailed in this dissertation which makes use of extracts taken from fiber samples. Lastly, we propose an instrumental setup coupling a microscope to a spectrofluorimeter for the purpose of taking EEM directly from a fiber sample. This setup makes use of the superior optics of the microscope for focusing excitation light onto the fiber sample. Initial studies have been performed on extracts from a single textile fiber and EEM collected from said fiber.
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Date Issued: 2009
Identifier: CFE0002833, ucf:48084
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0002833

Title: Advancements in Liquid Chromatography for the Determination of Polycyclic Aromatic Compounds in Environmental Samples.
Creator: Hayes, Hugh, Campiglia, Andres, Harper, James, Yestrebsky, Cherie, Frazer, Andrew, Coffey, Kevin, University of Central Florida
Abstract / Description: The purpose of this dissertation is to provide a new and improved analytical methodology for the analysis of polycyclic aromatic hydrocarbons (PAHs) and polycyclic aromatic sulfur heterocycles (PASHs) in environmental samples. Hundreds of PAHs and PASHs are present in the environment and have great toxicological importance and the chemical determination of these components is a priority. The complete normal-phase liquid chromatography (NPLC) retention behavior has been explored for these...
Show moreThe purpose of this dissertation is to provide a new and improved analytical methodology for the analysis of polycyclic aromatic hydrocarbons (PAHs) and polycyclic aromatic sulfur heterocycles (PASHs) in environmental samples. Hundreds of PAHs and PASHs are present in the environment and have great toxicological importance and the chemical determination of these components is a priority. The complete normal-phase liquid chromatography (NPLC) retention behavior has been explored for these compounds. Retention indices were determined for 124 PAHs, 62 methyl-PAHs (MePAHs), 67 PASHs, and 80 alkyl-PASHs on an aminopropyl (NH2) stationary phase which represents the most comprehensive study of polycyclic aromatic compounds in normal phase conditions to date. NPLC retention behavior for PAHs and PASHs directly correlated to the total number of aromatic carbons in the parent structures. The normal-phase retention behavior information was used to develop an NPLC fractionation procedure to aid in the sample cleanup for complex environmental matrices which can later be analyzed by gas chromatography/mass spectrometry (GC/MS). Standard Reference Material (SRM) 1597a (complex mixture of PAHs in coal tar), SRM 1991 (coal tar/petroleum extract), and SRM 1975 (diesel particulate extract) were analyzed before and after NPLC fractionation by using GC/MS. In SRM 1597a, the NPLC-GC/MS method allowed for the identification of 72 PAHs, 56 Me-PAHs, 35 PASHs, and 59 alkyl-PASHs. The NPLC-GC/MS procedure also provided the tentative identification of 74 PAHs and 117 MePAHs based on the molecular ion peak only. Furthermore, this method allowed for the following identification breakdown: SRM 1991(-)31 PASHs and 58 alkyl-PASHs; and SRM 1975(-)13 PASHs and 25 alkyl-PASHs.Additional work related to the NPLC fractionation of SRM 1597 included the collection of room-temperature fluorescence spectra for the 21 PAHs with molecular mass (MM) 302 Da known to be in SRM 1597a during reversed-phase liquid chromatography (RPLC) separation. Adding spectral features to a chromatographic run provided the tentative identification of 20 PAHs based on retention times and the presence of 18 were confirmed on the basis of spectral profiles. The advancements in liquid chromatography presented in this dissertation via NPLC fractionation along with RPLC stop-flow fluorescence spectra collection shows potential for becoming routine methodologies for PAC determination in complex environmental samples.
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Date Issued: 2018
Identifier: CFE0007190, ucf:52244
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0007190

Title: Photophysics of Organic Probes and their Applications in Bioimaging (&) Photodynamic Therapy.
Creator: Kim, Bosung, Belfield, Kevin, Zou, Shengli, Campiglia, Andres, Frazer, Andrew, Ali, Gul Shad, University of Central Florida
Abstract / Description: Over the past several decades the phenomenon of luminescence (divided into fluorescence and phosphorescence) has received great attention in the field of biological science. This quest has motivated scientists for a variety of applications, including fluorescence imaging. Fluorescence microscopy techniques that provide unique advantages, such as high spatial resolution and superior sensitivity, have been regarded as attractive tools in biophotonics. With the progress of ultrafast laser...
Show moreOver the past several decades the phenomenon of luminescence (divided into fluorescence and phosphorescence) has received great attention in the field of biological science. This quest has motivated scientists for a variety of applications, including fluorescence imaging. Fluorescence microscopy techniques that provide unique advantages, such as high spatial resolution and superior sensitivity, have been regarded as attractive tools in biophotonics. With the progress of ultrafast laser sources, two-photon absorption (2PA), in which a molecule absorbs two photons simultaneously, has opened possibilities of using it for various applications. Two-photon fluorescence microscopy (2PFM), which affords deeper tissue penetration and excellent three-dimensional (3D) images, is now being widely employed for bioimaging. This dissertation focuses on the design, synthesis, and photophysical characterization of new fluorophores, as well as desirable applications. Chapter 1 gives an account of a brief introduction of luminescence and 2PA, as well as their utilities in biological applications. In chapter 2, a series of new BODIPY derivatives are presented along with their comprehensive linear and nonlinear characteristics. They exhibited excellent photophysical properties including large extinction coefficients, high fluorescence quantum yields, good photostability, and reasonable two-photon absorption cross sections. Two promising compounds were further evaluated as NIR fluorescent probes in one-photon and two-photon fluorescence imaging. Chapter 3 provides the design, synthesis, and photophysical characterization of two BODIPY dyes. In order to assess the potential of using the dye as a fluorescent probe, Lysotracker Red, a commercial lysosomal marker, was investigated for comparison purposes. The results indicate that figure of merit of both compounds were three orders of magnitude higher than that of Lysotracker Red. With an eye towards applications, one of the compounds was encapsulated in silica-based nanoparticles for in vitro and ex vivo one-photon and two-photon fluorescence imaging, in which the surface of the nanoparticle was modified with RGD peptides for specific targeting. The nanoprobe exhibited good biocompatibility and highly selective RGD-mediated uptake in ?V?3 integrin-overexpressing cancers, while maintaining efficient fluorescence quantum yield and high photostability. In chapter 4, the synthesis and photophysical properties of a novel photosensitizer with heavy atoms (halogen) were presented. The dye exhibited low fluorescence quantum yield, resulting in high singlet oxygen generation quantum yield. In vitro photodynamic studies demonstrated that photosensitization of the agent can induce cellular damage, subsequently leading to cell death by a necrotic cell death mechanism, supporting the therapeutic potential of using the agent for photodynamic therapy.
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Date Issued: 2015
Identifier: CFE0006041, ucf:50977
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0006041

Title: Using Fluorescence to Characterize Four Day Simulated Distribution System Trihalomethane Content in Florida Groundwaters.
Creator: Ousley, Jonathan, Duranceau, Steven, Lee, Woo Hyoung, Sadmani, A H M Anwar, University of Central Florida
Abstract / Description: The United States Environmental Protection Agency (USEPA) regulates public water systems and has established limits for certain disinfection by products (DBPs) that have been linked to health effects, such as bladder cancer. The regulation of DBPs, specifically total trihalomethanes (TTHMs) and haloacetic acids (HAAs), have encouraged water treatment professionals to assess the type and amount of organic precursors in their supplies. Three of the more common water quality parameters that are...
Show moreThe United States Environmental Protection Agency (USEPA) regulates public water systems and has established limits for certain disinfection by products (DBPs) that have been linked to health effects, such as bladder cancer. The regulation of DBPs, specifically total trihalomethanes (TTHMs) and haloacetic acids (HAAs), have encouraged water treatment professionals to assess the type and amount of organic precursors in their supplies. Three of the more common water quality parameters that are monitored as DBP surrogates include dissolved organic carbon (DOC), ultraviolet absorbance (UV254), and specific ultraviolet absorbance (SUVA). Although DOC, UV254, and SUVA have been effectively correlated to DBP formation, efforts to correlate fluorescence excitation emission matrices (FEEM) to DBP formation remains limited within the drinking water community. In this research, a fluorescence regional integration (FRI) approach was used to compare FEEM with DOC, UV254, and SUVA as an alternative surrogate for characterizing TTHMs for groundwater sources located in south central Florida. To quantitatively evaluate FEEM, DOC, UV254, and SUVA as TTHM precursor surrogate parameters, a statistical correlation analysis was employed. Thirteen groundwater samples were collected from various Central Florida groundwater wells in Lake County, Polk County, and Palm Beach County, and analyzed for FEEM, DOC, UV254, and SUVA prior to determining the four-day TTHM concentration using a simulated distribution system dosing procedure. The FRI method was then used to quantify FEEM by dividing the three-dimensional matrix into five distinct regions, each representing a unique organic constituent. The volume under each region was determined and used for the correlation analysis.It was determined that a combinations of regions III and V of the FEEM possessed a strong linear correlation to four day TTHM content (R2 = 0.95) as compared to DOC (R2 = 0.906), UV254 (R2= 0.84), SUVA (R2 = 0.640), and the individual regions of the FEEM. However, DOC showed the strongest correlation when a second order polynomial regression was used (R2 = 0.937). Results for the individual regions of the FEEM revealed four day simulated TTHM correlation coefficients of 0.25, 0.62, 0.86, 0.74, and 0.88 for regions I through V respectively. These values indicated that a combination of regions III and V, which represent the fulvic and humic-like organic fractions detected by FEEM respectively, was the most accurate four day simulated TTHM precursor surrogate parameter based on the groundwater supplies tested. These results reveal that although DOC is still one of the strongest surrogate parameters to TTHM formation, fluorescence has also shown to also be a potentially strong surrogate for groundwaters. The implications of these results signify that fluorescence monitoring could be a viable method of measuring organic content in groundwaters once the technology further develops.
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Date Issued: 2016
Identifier: CFE0006839, ucf:51782
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0006839

Title: SYNTHESIS, CHARACTERIZATION, AND EVALUATION OF NEW REACTIVE TWO-PHOTON ABSORBING DYES FOR TWO-PHOTON EXCITED FLUORESCENCE IMAGING APPLICATIONS.
Creator: Hales, Katherine, Belfield, Kevin, University of Central Florida
Abstract / Description: Recent, cooperative advances in chemistry, computing, optics, and microelectronics have resulted in extraordinary developments in the biological sciences, resulting in the emergence of a novel area termed ¡¥biophotonics¡¦. The interdisciplinary nature of biophotonics cuts across virtually all disciplines, extending the frontiers of basic cellular, molecular, and biology research. This holds true for the development and application of the novel imaging modality utilizing...
Show moreRecent, cooperative advances in chemistry, computing, optics, and microelectronics have resulted in extraordinary developments in the biological sciences, resulting in the emergence of a novel area termed ¡¥biophotonics¡¦. The interdisciplinary nature of biophotonics cuts across virtually all disciplines, extending the frontiers of basic cellular, molecular, and biology research. This holds true for the development and application of the novel imaging modality utilizing multiphoton absorption and its extraordinary contribution to advances in bioimaging. Intimately involved in the revolution of nonlinear bioimaging has been the development of optical probes for probing biological function and activity. The focus of this dissertation is in the area of probe development, particularly ƒà-conjugated organic probes, optimized for efficient two-photon absorption followed by upconverted fluorescence for multiphoton bioimaging. Specifically, fluorene molecules, with enhanced two-photon absorbing (2PA) properties and high photostability, were prepared and characterized. Contemporary synthetic methods were utilized to prepare target fluorene derivatives expected to be highly fluorescent and, in particular, exhibit high two-photon absorptivity, suitable for two-photon excitation (2PE) fluorescence microscopy. The flexibility afforded through synthetic manipulation for integrating hydrophilic moieties into the fluorophore architecture to enhance compatibility with aqueous systems, more native to biological samples, was attempted. Incorporation of functional groups for direct covalent attachment onto biomolecules was also pursued to prepare fluorene derivatives as efficient 2PA reactive probes. Linear and two-photon spectroscopic characterizations on these novel compounds reveal they exhibit relatively high 2PA cross-sections on the order of ~100 GM units, which is greater than typical, commonly used fluorophores utilized in multiphoton bioimaging. Extensive photostability studies of representative fluorene compounds demonstrate these derivatives are photostable under one- and two-photon excitation conditions, exhibiting photodecomposition quantum yields on the order of 10-5. Additionally, preliminary cytotoxicity studies indicate these fluorene derivatives exhibit minimal cytotoxic effects on proliferating cells. Finally, their utility as high-performance, 2PA fluorescent probes in 2PE fluorescence microscopy imaging of biological samples was demonstrated in both fixed and live cells. Due to the low cytotoxicity, high photostability, efficient 2PA, and high fluorescence quantum yield, the probes were found suitable for relatively long-term, two-photon fluorescence imaging of live cells, representing a significant advance in biophotonics.
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Date Issued: 2005
Identifier: CFE0000685, ucf:46487
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0000685

Title: BIOPHYSICAL CHARACTERIZATION OF THE MEMBRANE BINDING DOMAIN OF THE PRO-APOPTOTIC PROTEIN BAX.
Creator: Garg, Pranav, Tatulian, Suren, University of Central Florida
Abstract / Description: The BCL-2 family of proteins tightly regulates the delicate balance between life and death. The pore forming Bax is a pro-apoptotic member belonging to this protein family. At the onset of apoptosis, monomeric cytoplasmic Bax translocates to the outer mitochondrial membrane, forms oligomeric pores thereby letting mitochondrial cytochrome c enter the cytosol and initiate the apoptotic cascade. The C-terminal "helix 9" is thought to mediate the membrane binding of BAX. A 20-amino acid peptide...
Show moreThe BCL-2 family of proteins tightly regulates the delicate balance between life and death. The pore forming Bax is a pro-apoptotic member belonging to this protein family. At the onset of apoptosis, monomeric cytoplasmic Bax translocates to the outer mitochondrial membrane, forms oligomeric pores thereby letting mitochondrial cytochrome c enter the cytosol and initiate the apoptotic cascade. The C-terminal "helix 9" is thought to mediate the membrane binding of BAX. A 20-amino acid peptide corresponding to Bax C-terminus (VTIFVAGVLTASLTIWKKMG) and two mutants where the two lysines are replaced with Glu (charge reversal mutant, EE) or Leu (charge neutralization mutant, LL) have been studied to elucidate the pore formation capabilities of Bax C-terminus and the underlying molecular mechanism. Interactions of the wild-type and the two mutant peptides with zwitterionic and anionic phospholipid membranes caused efficient membrane permeabilization, as documented by release of vesicle-entrapped fluorescent indicator calcein. Light scattering experiments showed that vesicles maintained their integrity upon peptide binding, indicating that the content leakage was due to pore formation and not vesicle degradation. Kinetics of calcein release at various peptide concentrations were used to determine the peptide-peptide association constants and the oligomeric state of the pore. The structure of membrane-bound peptides was analyzed by circular dichroism (CD) and attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy. CD data indicated all three peptides reconstituted in lipid vesicles contained [alpha]-helical and [beta]-strand structures. ATR-FTIR experiments indicated that the minimally hydrated samples of peptides in stacked lipid bilayers (absence of bulk water) were mostly [alpha]-helical but adopted mostly [beta]-sheet conformation in the presence of excess water. Finally, the depth of membrane insertion of the peptides was analyzed using tryptophan fluorescence quenching by dibromo-phosphatidylcholines brominated at various positions of their acyl chains. In case of zwitterionc phospholipid membranes, the single Trp16 was located at ~9 Å from membrane center. In case of membranes containing 30% of an anionic phospholipid, the depth of membrane insertion of the EE mutant was not affected but the wild-type and the LL mutant peptides were embedded much deeper into the membrane, with Trp16 located at 3-4 Å from membrane center. These results will help achieve a better understanding of the molecular mechanism of membrane pore formation of Bax protein. In addition, they provide insight into the molecular details of membrane pore formation by peptides and could facilitate the design and production of cytotoxic peptides with improved capabilities to lyse cells such as bacteria or cancer cells.
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Date Issued: 2011
Identifier: CFE0003983, ucf:48674
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0003983

Title: The Effect of Recycled Backwash Water Operations on Fouling in a Coagulation-Ultrafiltration Process and Impact of Preozonation on Membrane Productivity.
Creator: Biscardi, Paul, Duranceau, Steven, Sadmani, A H M Anwar, Lee, Woo Hyoung, Clausen, Christian, University of Central Florida
Abstract / Description: This dissertation consists of research that focused on pretreatment strategies to reduce fouling of ultrafiltration (UF) membranes used for drinking water treatment, and was segmented into four key components. (1) In the first component of the work, the long-term fouling behavior of a polyethersulfone (PES) hollow-fiber UF membrane was studied at the pilot-scale for treatment of surface water over a one-year period. Pilot testing of a coagulation-flocculation-sedimentation (CFS) pretreatment...
Show moreThis dissertation consists of research that focused on pretreatment strategies to reduce fouling of ultrafiltration (UF) membranes used for drinking water treatment, and was segmented into four key components. (1) In the first component of the work, the long-term fouling behavior of a polyethersulfone (PES) hollow-fiber UF membrane was studied at the pilot-scale for treatment of surface water over a one-year period. Pilot testing of a coagulation-flocculation-sedimentation (CFS) pretreatment system revealed that chemically irreversible fouling was poorly correlated with turbidity and total organic carbon. It was also shown that recycled backwash water may have impacted membrane process performance, and that chemically irreversible fouling was responsive to changes in pretreatment configuration. (2) In the second component, pre-oxidation with ozone (preozonation) was then studied as a pretreatment process to reduce natural organic matter (NOM) fouling at the pilot-scale. This work suggested that preozonation reduced long-term chemically irreversible fouling. The chemically reversible fouling index increased by 59%, indicating that preozonation changed the characteristics of the foulants, yielding more effective chemically enhanced backwashes. (3) Bench-scale work that studied changes in NOM characteristics associated with the improved process performance were performed using fluorescent excitation-emission (EEM) spectroscopy and high-performance size-exclusion chromatography (HPSEC). Specifically, ozone was applied prior to a CFS-UF process and compared to a CFS-UF condition without ozone as the control. Although CFS reduced turbidity by 29%, ozone, when integrated with CFS increased turbidity by 58%, impacting downstream UF performance. As expected, ozone, when integrated with CFS and UF reduced filtrate true color by 40%, UV254 absorbance by 11%, and SUVA by 30%, relative to the control, indicating that preozonation changed the characteristics of the dissolved organic carbon present in the source water. (4) Follow-up bench-scale research using fluorescent EEM spectroscopy and HPSEC assessed operational strategies that impacted organic fouling. Specifically, the fate of fluorescing substances during the recycling of membrane backwash water (MBWW) ahead of CFS-UF process was investigated. Bench-scale UF membranes were used to generate MBWW from a CFS-treated surface water containing 21 mg/L dissolved organic carbon (DOC) registering a 0.95 cm-1 UV254 absorbance that had been coagulated with 100 mg/L with polyaluminum chloride. CFS settled water, when processed with UF, produced MBWW containing 9 mg/L DOC registering a 0.25 cm-1 UV254 absorbance. HPSEC with UV254 detection demonstrated an analogous UV254 reduction as measured by detector response. However, fluorescence EEM spectroscopy revealed that protein-like substances, known to be associated with irreversible fouling, had been concentrated in the MBWW. In order to evaluate recycling operations on overall DOC removal in a CFS-UF process, a blend of 30% MBWW with 70% of raw water was treated, resulting in an overall DOC removal of 73%. However, without MBWW recycle, the CFS-UF process removed less of the influent DOC (63%). In summary, this research demonstrated that NOM characteristics within MBWW should be considered when recycling backwash water in PES membrane operations, and that preozonation reduces chemically irreversible fouling when incorporated into a CSF-UF system.
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Date Issued: 2016
Identifier: CFE0006074, ucf:50951
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0006074

Title: Solid Phase Extraction Room Temperature Fluorescence Spectroscopy for the Direct Quantification of Monohydroxy Metabolites of Polycyclic Aromatic Hydrocarbons in Urine Samples.
Creator: Calimag, Korina Jesusa, Campiglia, Andres, Belfield, Kevin, Yestrebsky, Cherie, Chumbimuni Torres, Karin, Schulte, Alfons, University of Central Florida
Abstract / Description: Polycyclic aromatic hydrocarbons (PAH) are important environmental pollutants originating from a wide variety of natural and anthropogenic sources. Because many of them are highly suspect as etiological agents in human cancer, chemical analysis of PAH is of great environmental and toxicological importance. Current methodology for PAH follows the classical pattern of sample preparation and chromatographic analysis. Sample preparation pre-concentrates PAH, simplifies matrix composition, and...
Show morePolycyclic aromatic hydrocarbons (PAH) are important environmental pollutants originating from a wide variety of natural and anthropogenic sources. Because many of them are highly suspect as etiological agents in human cancer, chemical analysis of PAH is of great environmental and toxicological importance. Current methodology for PAH follows the classical pattern of sample preparation and chromatographic analysis. Sample preparation pre-concentrates PAH, simplifies matrix composition, and facilitates analytical resolution in the chromatographic column. Among the several approaches that exist to pre-concentrate PAH from water samples, the Environmental Protection Agency (EPA) recommends the use of solid-phase extraction (SPE). High-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS) are the basis for standard PAH identification and determination. Ultraviolet (UV) absorption and room temperature fluorescence detection are both widely used in HPLC, but the specificity of these detectors is modest. Since PAH identifi(&)#172;cation is solely based on retention times, unambiguous PAH identification requires complete chromatographic resolution of sample components. When HPLC is applied to (")unfamiliar(") samples, the EPA recommends that a supporting analytical technique such as GC-MS be applied to verify compound identification and to check peak-purity HPLC fractions. Independent of the volume of extracted water, the approximate time required to separate and determine the sixteen (")priority pollutants(") (EPA-PAH) via HPLC is approximately 60min. If additional GC-MS analysis is required for unambiguous PAH determination, the total analysis time will reach 2-3 hours per sample. If the concentrations of target species are found to lie outside the detector's response range, the sample must be diluted and the process repeated. These are important considerations when routine analysis of numerous samples is contemplated. Parent PAH are relatively inert and need metabolic activation to express their carcinogenicity. By virtue of the rich heterogeneous distribution of metabolic products they produce, PAH provide a full spectrum of the complexity associated with understanding the initial phase of carcinogenesis. PAH metabolites include a variety of products such as expoxides, hydroxyl aromatics, quinines, dihydrodiols, dioepoxides, tetrols and water soluble conjugates. During the past decades tremendous efforts have been made to develop bio-analytical techniques that possess the selectivity and sensitivity for the problem at hand. Depending on the complexity of the sample and the relative concentrations of the targeted metabolites, a combination of sample preparation techniques is often necessary to reach the limits of detection of the instrumental method of analysis. The numerous preparation steps open ample opportunity to metabolite loss and collection of inaccurate data. Separation of metabolites has been accomplished via HPLC, capillary electrophoresis (CE) and GC-MS. Unfortunately, the existence of chemically related metabolic products with virtually identical fragmentation patterns often challenges the specificity of these techniques. This dissertation presents significant improvements in various fronts. Its first original component (-) which we have named solid-phase nano-extraction (SPNE) - deals with the use of gold nanoparticles (Au NPs) as extracting material for PAH. The advantages of SPNE are demonstrated for the analysis of PAH in water samples via both HPLC1 and Laser-Excited Time-Resolved Shpol'skii Spectroscopy (LETRSS).2 The same concept is then extended to the analysis of monohydroxy-PAH in urine samples via SPE- HPLC3 and In-Capillary SPNE-CE.4 The second original component of this dissertation describes the application of Shpol'skii Spectroscopy to the analysis of polar PAH metabolites. The outstanding selectivity and sensitivity for the direct analysis of PAH at trace concentration levels has made Shpol'skii spectroscopy a leading technique in environmental analysis.5 Unfortunately, the requirement of a specific guest-host combination - typically a non-polar PAH dissolved in an n-alkane - has hindered its widespread application to the field of analytical chemistry. This dissertation takes the first steps in removing this limitation demonstrating its feasibility for the analysis of polar benzo[a]pyrene metabolites in alcohol matrixes.
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Date Issued: 2013
Identifier: CFE0005141, ucf:50693
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0005141

Title: Application of Two-Photon Absorbing Fluorene-Containing Compounds in Bioimaging and Photodyanimc Therapy.
Creator: Yue, Xiling, Belfield, Kevin, Campiglia, Andres, Miles, Delbert, Frazer, Andrew, Cheng, Zixi, University of Central Florida
Abstract / Description: Two-photon absorbing (2PA) materials has been widely studied for their highly localized excitation and nonlinear excitation efficiency. Application of 2PA materials includes fluorescence imaging, microfabrication, 3D data storage, photodynamic therapy, etc. Many materials have good 2PA photophysical properties, among which, the fluorenyl structure and its derivatives have attracted attention with their high 2PA cross-section and high fluorescence quantum yield.Herein, several compounds with...
Show moreTwo-photon absorbing (2PA) materials has been widely studied for their highly localized excitation and nonlinear excitation efficiency. Application of 2PA materials includes fluorescence imaging, microfabrication, 3D data storage, photodynamic therapy, etc. Many materials have good 2PA photophysical properties, among which, the fluorenyl structure and its derivatives have attracted attention with their high 2PA cross-section and high fluorescence quantum yield.Herein, several compounds with 2PA properties are discussed. All of these compounds contain one or two fluorenyl core units as part of the conjugated system. The aim of this dissertation is to discuss the application of these compounds according to their photophysical properties. In chapters 2 to 4, compounds were investigated for cell imaging and tissue imaging. In chapter 5, compounds were evaluated for photodynamic therapy effects on cancer cells. Chapters 2 and 3 detail compounds with quinolizinium and pyran as core structures, respectively. Fluorene was introduced into structures as substituents. Quinolizinium structures exhibited a large increase in fluorescence when binding with Bovine Serum Albumin (BSA). Further experiments in cell imaging demonstrated a fluorescence turn-on effect in cell membranes, indicating the possibility for these novel compounds to be promising membrane probes. Pyran structures were conjugated with arginylglycylaspartic acid peptide (RGD) to recognize integrin and introduced in cells and an animal model with tumors. Both probes showed specific targeting of tumor vasculature. Imaging reached penetration as deep as 350 ?m in solid tumors and exhibited good resolution. These results suggest the RGD-conjugated pyran structure should be a good candidate probe for live tissue imaging. Chapter 4 applied a fluorene core structure conjugated with RGD as well. Application of this fluorenyl probe compound is in wound healing animal models. Fluorescence was collected from vasculature and fibroblasts up to ? 1600 ?m within wound tissue in lesions made on the skin of mice. The resolution of images is also high enough to recognize cell types by immunohistochemical staining. This technology can be applied for reliable quantification and illustration of key biological processes taking place during tissue regeneration in the skin. Chapter 5 describes three fluorenyl core structures with photoacid generation properties. One of the structures showed excellent photo-induced toxicity. Cancer cells underwent necrotic cell death due to pH decrease in lysosomes and endosomes, suggesting a new mechanism for photodynamic therapy.
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Date Issued: 2014
Identifier: CFE0005565, ucf:50276
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0005565

Title: Zinc Sulfide:manganese doped Quantum rods for detection of metal ions and a business model for future sales.
Creator: Teblum, Andrew, Santra, Swadeshmukul, Gesquiere, Andre, Soskin, Mark, University of Central Florida
Abstract / Description: Hexavalent chromium is an extremely carcinogenic chemical that has been widely produced in the United States. This has led to major waste contamination and pollution throughout the country. According to the Environmental Working Group Hexavalent chromium has been found in 89% of city tap water. Most people believe they are safe using regular home filter systems however that is not true. A more expensive ion exchange water treatment unit is required. Therefore to protect yourselves from this...
Show moreHexavalent chromium is an extremely carcinogenic chemical that has been widely produced in the United States. This has led to major waste contamination and pollution throughout the country. According to the Environmental Working Group Hexavalent chromium has been found in 89% of city tap water. Most people believe they are safe using regular home filter systems however that is not true. A more expensive ion exchange water treatment unit is required. Therefore to protect yourselves from this carcinogenic metal a reliable test is required. In this study we have developed a Zinc Sulfide Manganese doped Quantum Rod technology to detect for presence of chromate and other harmful transitional metals in drinking water. Quantum Rods were synthesized using a hydrothermal reaction method. They were fully characterized using UV-visible absorption spectroscopy, fluorescence emission spectroscopy, X-ray Photoelectric Spectroscopy (XPS) and High Resolution Transmission Electron Microscopy (HRTEM). Quantum Rod metal detection studies were done with 28 different ions in a 96-well fluorescent plate reader. Results show that highest sensitivity to 8 ions including the toxic ions of chromate and mercury allowing us to create a sensor to detect these items.
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Date Issued: 2014
Identifier: CFE0005268, ucf:50569
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0005268

Title: Dissertation Title: Development of molecular and cellular imaging tools to evaluate gene and cell based therapeutic strategies in vivo.
Creator: Xia, Jixiang, Ebert, Steven, Khaled, Annette, Cheng, Zixi, Daniell, Henry, University of Central Florida
Abstract / Description: Molecular imaging modalities are important tools to evaluate the efficacy of gene delivery systems and cell-based therapies. Development and application of these modalities will advance our understanding of the mechanism of transgene expression and cell fate and functions. Physical gene transfer methods hold many advantages over viral vectors among gene therapeutic strategies. Here, we evaluated the efficacy of biolistic ((")gene gun(")) gene targeting to tissues with non-invasive...
Show moreMolecular imaging modalities are important tools to evaluate the efficacy of gene delivery systems and cell-based therapies. Development and application of these modalities will advance our understanding of the mechanism of transgene expression and cell fate and functions. Physical gene transfer methods hold many advantages over viral vectors among gene therapeutic strategies. Here, we evaluated the efficacy of biolistic ((")gene gun(")) gene targeting to tissues with non-invasive bioluminescence imaging (BLI) methods. Plasmids carrying the firefly luciferase reporter gene were transfected into mouse skin and liver using biolistics, and BLI was measured at various time points after transfer. With optimized DNA loading ratio (DLRs), reporter gene expression reached to peak 1day after transfer to mouse skin, and the maximum depth of tissue penetration was between 200-300?m. Similar peak expression of reporter gene was found in mouse liver but the expression was relatively stable 4-8 days post-biolistic gene transfer and remained for up to two weeks afterward. Our results demonstrated BLI was an efficient strategy for evaluation of reporter gene expression in the same animals over a period of up to two weeks in vivo. Different tissues showed different expression kinetics, suggesting that this is an important parameter to consider when developing gene therapy strategies for different target tissues. We also employed BLI to measure differentiation of mouse embryonic stem (ES) cells into beating cardiomyocytes in vitro and in vivo. A subset of these cardiomyocytes appears to be derived from an adrenergic lineage that ultimately contribute to substantial numbers of cardiomyocytes primarily on the left side of the heart. At present, it is unclear what the precise role of these cardiac adrenergic cells is with respect to heart development, though it is known that adrenergic hormones (adrenaline and noradrenaline) are essential for embryonic development since mice lacking them die from apparent heart failure during the prenatal period. To identify and characterize cardiac adrenergic cells, we developed a novel mouse genetic model in which the nuclear-localized enhanced green fluorescent protein (nEGFP) reporter gene was targeted to the first exon of the Phenylethanoamine N-transferase (Pnmt) gene, which encodes for the enzyme that converts noradrenaline to adrenaline, and hence serves as a marker for adrenergic cells. Our results demonstrate this knock-in strategy effectively marked adrenergic cells in both fetal and adult mice. Expression of nEGFP was found in Pnmt-positive cells of the adult adrenal medulla, as expected. Pnmt-nEGFP expression also recapitulated endogenous Pnmt expression in the embryonic mouse heart. In addition, nEGFP and Pnmt expression were induced in parallel during differentiation of pluripotent mouse ES cells into beating cardiomyocytes. This new mouse genetic model provides a useful new tool for studying the properties of adrenergic cells in different tissues. We also identified two limitations of the Pnmt-nEGFP model. One is that the amount of nEGFP expressed within individual adrenergic cells was highly variable. Secondly, expression of nEGFP in the embryonic heart was of low abundance and difficult to distinguish from background autofluorescence. To overcome these limitations, we developed two alternative genetic models to investigate adrenergic cells: (1) Mouse embryonic stem cells, which have been previously targeted with Pnmt-Cre recombinase gene, were additionally targeted with a dual reporter plasmid which covered both a loxP-flanked cDNA of red fluorescence protein (HcRed) and also EGFP. Under the undifferentiated status, cells emit red fluorescence as transcription stops before EGFP coding sequence. After differentiation into beating cardiomyoctyes, some cells switch fluorescence from red to green, indicating that excision of loxP-flanked sequences by Cre since Pnmt had been activated. (2) A surface marker, truncated low-affinity nerve growth factor receptor (?LNGFR) was used as the reporter gene as cells expressing this marker can be enriched by magnetic-activated cell sorting (MACS), a potentially efficient way to yield highly purified positive cells at low input abundance in a population. Through a series of subcloning steps, the targeting construct, Pnmt-?LNGFR-Neo-DTA was created and electroporated into 7AC5EYFP embryonic stem cells. Correctly targeted cells were selected by positive and negative screening. These cells provide a new tool with which to identify, isolate, and characterize the function of adrenergic cells in the developing heart, adrenal gland, and other tissues where adrenergic cells make important contributions.
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Date Issued: 2011
Identifier: CFE0004491, ucf:49287
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0004491

Title: THREE-PHOTON ABSORPTION PROCESS IN ORGANIC DYES ENHANCED BY SURFACE PLASMON RESONANCE.
Creator: Cohanoschi, Ion, Hernandez, Florencio, University of Central Florida
Abstract / Description: Multi-photon absorption processes have received significant attention from the scientific community during the last decade, mainly because of their potential applications in optical limiting, data storage and biomedical fields. Perhaps, one of the most investigated processes studied so far has been two-photon absorption (2PA). These investigations have resulted in successful applications in all the fields mentioned above. However, 2PA present some limitations in the biomedical field when...
Show moreMulti-photon absorption processes have received significant attention from the scientific community during the last decade, mainly because of their potential applications in optical limiting, data storage and biomedical fields. Perhaps, one of the most investigated processes studied so far has been two-photon absorption (2PA). These investigations have resulted in successful applications in all the fields mentioned above. However, 2PA present some limitations in the biomedical field when pumping at typical 2PA wavelengths. In order to overcome these limitations, three-photon absorption (3PA) process has been proposed. However, 3PA in organic molecules has a disadvantage, typical values of σ3' are small (10-81 cm6s2/photon2), therefore, 3PA excitation requires high irradiances to induce the promotion of electrons from the ground state to the final excited state. To overcome this obstacle, specific molecules that exhibit large 3PA cross-section must be designed. Thus far, there is a lack of systematic studies that correlate 3PA processes with the molecular structure of organic compounds. In order to fill the existent gap in 3PA molecular engineering, in this dissertation we have investigated the structure/property relationship for a new family of fluorene derivatives with very high three-photon absorption cross-sections. We demonstrated that the symmetric intramolecular charge transfer as well as the -electron conjugation length enhances the 3PA cross-section of fluorene derivatives. In addition, we showed that the withdrawing electron character of the attractor groups in a pull-pull geometry proved greater 3PA cross-section. After looking for alternative ways to enhance the effective σ3' of organic molecules, we investigated the enhancement of two- and three-photon absorption processes by means of Surface Plasmon. We demonstrated an enhancement of the effective two- and three-photon absorption cross-section of an organic compound of 480 and 30 folds, respectively. We proved that the enhancement is a direct consequence of the electric field enhancement at a metal/buffer interface. Next, motivated by the demands for new materials with enhanced nonlinear optical properties, we studied the 3PA of Hematoporphyrin IX and J-aggregate supramolecular systems. As a result, we were able to propose the use of 3PA in photodynamic therapy using Photofrin, the only drug approved by the FDA for PDT.
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Date Issued: 2006
Identifier: CFE0001362, ucf:46981
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0001362

Title: UP-CONVERSION IN RARE-EARTH DOPED MICRO-PARTICLES APPLIED TO NEW EMISSIVE 2D DISLAYS.
Creator: Milliez, Anne, Bass, Michael, University of Central Florida
Abstract / Description: Up-conversion (UC) in rare-earth co-doped fluorides to convert diode laser light in the near infrared to red, green and blue visible light is applied to make possible high performance emissive displays. The infrared-to-visible UC in the materials we study is a sequential form of non-linear two photon absorption in which a strong absorbing constituent absorbs two low energy photons and transfers this energy to another constituent which emits visible light. Some of the UC emitters' most...
Show moreUp-conversion (UC) in rare-earth co-doped fluorides to convert diode laser light in the near infrared to red, green and blue visible light is applied to make possible high performance emissive displays. The infrared-to-visible UC in the materials we study is a sequential form of non-linear two photon absorption in which a strong absorbing constituent absorbs two low energy photons and transfers this energy to another constituent which emits visible light. Some of the UC emitters' most appealing characteristics for displays are: a wide color gamut with very saturated colors, very high brightness operation without damage to the emitters, long lifetimes and efficiencies comparable to those of existing technologies. Other advantages include simplicity of fabrication, versatility of operating modes, and the potential for greatly reduced display weight and depth. Thanks to recent advances in material science and diode laser technology at the excitation wavelength, UC selected materials can be very efficient visible emitters. However, optimal UC efficiencies strongly depend on chosing proper operating conditions. In this thesis, we studied the conditions required for optimization. We demonstrated that high efficiency UC depends on high pump irradiance, low temperature and low scattering. With this understanding we can predict how to optimally use UC emitters in a wide range of applications. In particular, we showed how our very efficient UC emitters can be applied to make full color displays and very efficient white light sources.
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Date Issued: 2006
Identifier: CFE0001058, ucf:46828
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0001058

Title: SQUARAINE DYES FOR TWO-PHOTON FLUORESCENCE BIOIMAGING APPLICATIONS.
Creator: Colon Gomez, Maria, Belfield, Kevin, University of Central Florida
Abstract / Description: Near-infrared emitting squaraine dyes are promising candidates for bioimaging applications. Two-photon fluorescence microscopy (2PFM) imaging is a powerful tool being used for studying biological function since it produces 3D images with minimal damage to cells and lower fluorophore photobleaching. The fluorescence wavelength of squaraine dyes normally falls in the near infrared region, providing deeper penetration through biological samples such as thick tissue sections. Squaraine dyes that...
Show moreNear-infrared emitting squaraine dyes are promising candidates for bioimaging applications. Two-photon fluorescence microscopy (2PFM) imaging is a powerful tool being used for studying biological function since it produces 3D images with minimal damage to cells and lower fluorophore photobleaching. The fluorescence wavelength of squaraine dyes normally falls in the near infrared region, providing deeper penetration through biological samples such as thick tissue sections. Squaraine dyes that could work for imaging cells and tissues for 2PFM imaging were synthesized and underwent comprehensive photophysical characterization, such as UV-Vis absorption, fluorescence, and anisotropy. The squaraine dyes were tested for cell toxicity to determine the concentration at which the cells should be incubated with the dye for 2PFM. In addition, the squaraine dyes were incubated with cancer cells to evaluate their utility in the bioimaging process. The squaraine dye that is not soluble in water can be incorporated in silica nanoparticles or micelles to facilitate dispersal in water for evaluation of its use as a probe. The prospective squaraine dyes can be used in cells and tissues for imaging that can then be analyzed to ascertain its use as a probe for biomedical applications, such as early cancer detection.
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Date Issued: 2013
Identifier: CFH0004338, ucf:45020
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFH0004338

Title: UNDERSTANDING THE ROLE OF A HEMERYTHRIN-LIKE PROTEIN IN MYCOBACTERIUM TUBERCULOSIS.
Creator: Herndon, Caitlyn, Rohde, Kyle, University of Central Florida
Abstract / Description: According to the Centers for Disease Control and Prevention (CDC), 8 million people each year are infected with Mycobacterium tuberculosis (Mtb) leading to 1.5 million deaths annually. This staggering number calls for advancements in understanding this bacterium so progress can be made in treating and preventing the disease. It is particularly important to understand mechanisms by which TB survives inside hostile host immune cells known as macrophages and within hypoxic granuloma lesions of...
Show moreAccording to the Centers for Disease Control and Prevention (CDC), 8 million people each year are infected with Mycobacterium tuberculosis (Mtb) leading to 1.5 million deaths annually. This staggering number calls for advancements in understanding this bacterium so progress can be made in treating and preventing the disease. It is particularly important to understand mechanisms by which TB survives inside hostile host immune cells known as macrophages and within hypoxic granuloma lesions of the lung. Preliminary microarray data has shown that a TB gene known as Rv2633c is induced upon macrophage invasion. Bioinformatic analysis of Rv2633c coding sequence shows the product of Rv2633c has homology with hemerythrin-like proteins. Hemerythrins are a class of proteins commonly used to bind oxygen and sense nitric oxide and iron, leading us to hypothesize a role for Rv2633c in surviving hypoxic or nitrosative stress encountered within macrophages and granulomas. My first aim will be to generate a reporter strain of Mycobacterium smegmatis (Msm) expressing the mCherry fluorescent protein driven by the Rv2633c promoter. This tool will allow us to determine the stress conditions (i.e. hypoxia, nitric oxide treatment, acid pH) that activate expression of this gene by measuring the change in fluorescence. Linking the regulation of Rv2633c to specific environmental cues relevant to infections in vivo will provide insight into the role of this unique protein. Secondly, a knockout mutant of Rv2633c in the attenuated M. bovis BCG will be constructed and characterized to determine the importance and function of this protein during TB infections.
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Date Issued: 2014
Identifier: CFH0004647, ucf:45291
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFH0004647

fluorescence (10)	+ -
Fluorescence (6)	+ -
two-photon absorption (6)	+ -
Two-photon absorption (3)	+ -
bioimaging (3)	+ -

fluorescence microscopy (3)	+ -
fluorescence spectroscopy (3)	+ -
Circular Dichroism (2)	+ -
Fluorescence Spectroscopy (2)	+ -
M-PFC (2)	+ -
Mycobacterium tuberculosis (2)	+ -
Shpol'skii spectroscopy (2)	+ -
TB (2)	+ -
apoptosis (2)	+ -
luminescence (2)	+ -
optical data storage (2)	+ -
photostability (2)	+ -
polycyclic aromatic hydrocarbons (2)	+ -
stimulated emission depletion (2)	+ -
tuberculosis (2)	+ -
two-photon fluorescence microscopy (2)	+ -
1 (1)	+ -
1JCC (1)	+ -
2 (1)	+ -
3-triazole derivative (1)	+ -
5-methylmellein (1)	+ -
AP-1 (1)	+ -
ATR-FTIR (1)	+ -
Adherent-invasive Escherichia coli (1)	+ -
Analytical Chemistry (1)	+ -

ETD Collection (39)	+ -
Honors Collection (5)	+ -

University of Central Florida (44)	+ -
Belfield, Kevin (15)	+ -
Campiglia, Andres (14)	+ -
Cheng, Zixi (8)	+ -
Frazer, Andrew (6)	+ -

Zou, Shengli (6)	+ -
Chumbimuni Torres, Karin (5)	+ -
Harper, James (5)	+ -
Lee, Woo Hyoung (4)	+ -
Miles, Delbert (3)	+ -
Rex, Matthew (3)	+ -
Rohde, Kyle (3)	+ -
Bass, Michael (2)	+ -
Beazley, Melanie (2)	+ -
Bhattacharya, Aniket (2)	+ -
Clausen, Christian (2)	+ -
Duranceau, Steven (2)	+ -
Gesquiere, Andre (2)	+ -
Hernandez, Florencio (2)	+ -
Khaled, Annette (2)	+ -
Sadmani, A H M Anwar (2)	+ -
Santra, Swadeshmukul (2)	+ -
Schulte, Alfons (2)	+ -
Sigman, Michael (2)	+ -
Tatulian, Suren (2)	+ -
Teter, Kenneth (2)	+ -
Yestrebsky, Cherie (2)	+ -
Zhai, Lei (2)	+ -
Alcedo, Karel (1)	+ -
Ali, Gul Shad (1)	+ -

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