A set of three through-bond energy transfer cassettes based on BODIPY as a donor and cyanine dyes as acceptors has been prepared via Sonogashira couplings, and their photophysical properties were examined. These cassettes fluoresce around 600 to 800 nm and are resolved by approximately 100 nm. This property is an important factor for multiplexing study in cellular imaging. Several useful fluorescent probes such as 5- and 6-carboxyfluorescein, water-soluble BODIPY, and water-soluble Nile Blue dyes, have also been synthesized and their photophysical properties studied. We have also attempted to develop a method for protein mono-labeling via a solidphase approach. The labeling of protein with one fluorescent dye facilitates quantification and single molecule imaging in biological applications. Various solidsupports such as PEGA, CPG, and BSA-coated CPG, were tested. Photolabile and chemically cleavable linkers were prepared to connect solid-supports and fluorophores. Unfortunately, our approach to the fluorescent mono-labeling of native proteins did not give us any conclusive results.

No description available.

The importance of sensing Nitric Oxide (NO) in physiology and medicine has led us to explore the reactivity of NO with organometallic Re dyes. Rhenium complexes were synthesized with the ability to react with NO and sense it under physiological conditions. Fluorescent 1,10-phenantroline complexes (phen)Re(PPh3)(CO)2OSO2CF3 (1) and (phen)Re(CH3CN)(CO)2OSO2CF3 (3) can sense NO in the range of 10 - 150 mM showing a decrease in fluorescence response at 514 nm and 532 nm respectively, upon NO-donor addition (lexc = 360 nm). (phen)Re(THF)(CO)2OSO2CF3 (2) showed a ratiometric response with a decrease in fluorescence intensity at 585 nm and an increase at 445 nm. These complexes were characterized by FT-IR, 1H-NMR, 13C-NMR, and 19F‑NMR. The quantum yields (F) and lifetimes (t) were also determined. On the basis of 19F‑NMR and 1H-NMR spectroscopic evidence, the proposed mechanism of action involves dissociation of CF3SO3- (abbreviated as OTf-) together with PPh3, (for 1), THF (for 2), or CH3CN (for 3). Fluorescence microscopy showed that 1 permeates through the cell membrane of rat aortic endothelial cells (RAOEC) and 2 gives increase in cell fluorescence response at 450 nm when exogenous NO is added to RAOEC. The Re complexes of dppz (dppz = 2,2’-dipyrido[3,2-a:2,3’-c]phenazine) (dppz)Re(THF)(CO)2OSO2CF3 (A2) and (dppz)Re(CH3CN)(CO)2OSO2CF3) (A3) were synthesized using the same methods as for complexes 1 and 3. Even though the dppz complexes exhibited similar behavior than the phenanthroline analogs, the signal changes were small and the results were inconclusive. The a-Diimine Re complexes of Xy-DAD (XyDAD = 1,4-bis-[2,6-dimethylphenyl]-1,4-diazo-1,3-butadiene) (Xy-DAD)Re(THF)(CO)2OSO2CF3 (8) and (Xy-DAD)Re(CH3CN)(CO)2OSO2CF3 (9) showed reactivity with NO and CN- with a response in the visible range of the absorption spectrum. These diazadiene (DAD) complexes showed decreased reactivity with NO and CN- in the presence of oxidants. The i-PrDAD complexes (i-PrDAD = 1,4‑bis-[2,6-diisopropylphenyl]-1,4-diazo-1,3-butadiene) (i‑Pr‑DAD)Re(THF)(CO)2OTf (10) and (i‑Pr‑DAD)Re(CH3CN)(CO)2OTf (11) exhibited similar behavior with the Xy-DAD analogs, but the spectral changes were much smaller and the results were inconclusive. The ESI-MS Mass Spectrometry studies of the phenanthroline complexes 1, and 2, after reaction with NO-donor showed that 1 and 2 react with NO to give [Re(CO)2(NO)2S2]+ species, while (phen)Re(CO)3Cl and (phen)Re(CO)3OSO2CF3 do not show any metal-NO containing products, which is consistent with the fluorescence studies.

Yes / Ex vivo wounded human skin organ culture is an invaluable tool for translationally relevant preclinical wound healing research. However, studies incorporating this system are still underutilized within the field because of the low throughput of histological analysis required for downstream assessment. In this study, we use intravital fluorescent dye to lineage trace epidermal cells, demonstrating that wound re‐epithelialization of human ex vivo wounds occurs consistent with an extending shield mechanism of collective migration. Moreover, we also report a relatively simple method to investigate global epithelial closure of explants in culture using daily fluorescent dye treatment and en face imaging. This study is the first to quantify healing of ex vivo wounds in a longitudinal manner, providing global assessments for re‐epithelialization and tissue contraction. We show that this approach can identify alterations to healing with a known healing promoter. This methodological study highlights the utility of human ex vivo wounds in enhancing our understanding of mechanisms of human skin repair and in evaluating novel therapies to improve healing outcome. / University of Manchester Strategic Fund; Wellcome Trust; BBSRC; Ministry of Higher Education, Malaysia Universiti; Sains Malaysia

Near-level Prairie landscapes have received limited attention in hydrological research. For this thesis, hydrometric measurements and four tracing experiments were completed at three “riparian-to-stream” sites in the Catfish Creek Watershed (southeastern Manitoba) to enhance Prairie hydrology understanding. First, hydrologic state variables were examined to infer vertical and lateral water movement. Second, tracer data were analyzed to evaluate the relative importance of surface versus subsurface water movement. Results show that hydrologic state variables can be useful for inferring riparian-to-stream water movement. Tracer data also revealed that subsurface water movement can contribute significantly to streamflow during snowmelt- and rainfall-triggered events in the study watershed. This thesis demonstrated that subsurface flow is a significant runoff generation mechanism in Prairie landscapes, thus challenging surface water-focused conceptualizations and management strategies that are traditionally used. The findings summarized in this thesis will be critical to improve the performance of hydrological models when applied to the Prairies. / February 2017

A challenge exists for understanding the origin of color for structurally colored, 3-dimensional bioorganic nanostructures, such as the scales of butterflies, beetles, and moths. Complex, hierarchical structures found within such scales create the overall scale appearance. The controlled alteration of color through material deposition and the addition of new optical functionalities to such structures are other areas of scientific interest. This dissertation addresses these challenges with a first-of-its-kind, systematic isolation (deconstruction) of scale component nanostructures, followed by evaluation of optical property/structure correlations. The additive deposition (constructive alteration) of emissive materials to structurally-colored templates complements this deconstructive approach towards understanding the origin of color in butterfly scales. Discoveries made through this work may help advance the bioinspired design of synthetic optical structures and subsequent color control through the addition of multilayered, emissive optical components.

This master's thesis deals with the ways of cell viability determination and its measuring. The first part of this thesis has a brief introduction into the theory of cell viability, also the methods of cell viability measurement and different types of fluorescent probes are described. The thesis further includes a brief description of the image processing methods, as well as the fluorescent microscope and applied filters are described. In the practical part the system implemented at a graphical programming environment LabVIEW used for the cell viability evaluation is described, functions of applied units are explained. The program is checked on model data.

4′,6-Diamidine-2-phenylindole (DAPI) is a common fluorochrome that is able to bind to deoxyribonucleic acid (DNA) with distinct, sequence-dependent enhancement of fluorescence. This work presents the synthesis of a new multifunctional compound that includes the fluorescent dye as a ⁹⁹ᵐTechnetium (⁹⁹ᵐTc) carrier. A new technique for the bioconjugation of DAPI with 6-hydrazinonicotinic acid (HYNIC) through an amide linkage was developed. The radiolabelling was performed with HYNIC as a chelator and N-IJ2-hydroxy-1,1-bisIJhydroxymethyl)ethyl)glycine (tricine) as a coligand. Furthermore, experimental evidence showed that ⁹⁹ᵐTc complexes with DAPI as DNA-binding moieties are detectable in living Fischer rat thyroid follicular cell line 5 (FRTL5) and their nuclei. The investigations indicated further that the new HYNIC-DAPI derivative is able to interact with double-stranded DNA. This establishes the possibility of locating ⁹⁹ᵐTc in close proximity to biological structures of living cells, of which especially the genetic information-carrying cell compartments are at the centre of interest. In this context, further investigations are related to the radiotoxic effects of DNA-bound ⁹⁹ᵐTc-HYNIC-DAPI derivatives and dosimetric calculations.

The rapid and accurate identification of the bacterium of the genus Lactobacillus, which are an important part of the normal gastrointestinal microflora and fermented dairy products are currently mainly used amplification methods. The aim of the study was to analyze the possibility of resolution of selected bacterial strains of the genus Lactobacillus, using the metod of polymerase chain reaction in the real time combined with high resolution melting curve analysis (qPCR HRM). It was tested five primers designed for qPCR-HRM analysis of lactic acid bacteria. The specificity of the primers was also verified simultaneously using bioinformatic analysis. On the basis of analysis of the DNA were selected as the most appropriate primers P1V1/P2V1, V3F/V3R and V6F/V6R. The suitability of the primers V3F/V3R and V6F/V6R was verified on a complex sample of food supplement from which the DNA was isolated using magnetic particles. The presence of bacteria of the genus Lactobacillus was performed using high resoluting melting analysis curves. The obtained results were in agreement with the information given by the manufacturer.