Fluorescence microscopy is a powerful analytical tool for visualizing biological processes at the subcellular level. In this regard, 1,3,5-triarylpyrazoline based fluorescent probes which act as "turn-on" probes, have been extensively researched. These probes achieve their fluorescence "turn-on" response by inhibition of fluorescence quenching by acceptor-excited photoinduced electron transfer upon binding of an analyte. It has been recently shown that some fluorescent probes used in biological research form colloids composed of nanoparticles, due to their hydrophobic character. This hydrophobic character can also lead to partitioning of the probe into cellular membranes. Colloid formation and membrane partitioning may affect the probes' photophysical properties such as absorption and emission wavelength and quantum yields. Recently, a series of 1,3,5-triarylpyrazolines synthesized in our group by M. T. Morgan, showed no formation of aggregates in aqueous buffer. Surprisingly, these probes increased their fluorescence intensity in the presence of liposomes. The photoinduced electron transfer process is greatly affected by the polarity of the medium in which the probe is used. In this study, the effect of membrane proximity on the photoinduced electron transfer process for pyrazoline based "turn-on" probes has been investigated. A series of water soluble 1,3,5-triarylpyrazolines have been synthesized in which a N,N-dialkylaniline moiety acts as an electron donor and a proton acceptor and an alkylated sulfonamide moiety acts as a molecular anchor for interaction with neutral and anionic liposomes.

<p>The aim of this diploma work is to improve selectivity and sensitivity in DNA-chips by utilizing fluorescence resonance energy transfer (FRET) between conjugated polyelectrolytes (CPEs) and fluorophores.</p><p>Leclerc and co-workers have presented successful results from studies of super FRET between fluorophore tagged DNA and a CPE during hybridisation of the double strand. Orwar and co-workers have constructed a DNA-chip using standard photo lithography creating a pattern of the hydrophobic photoresist SU-8 and cholesterol tagged DNA (chol-DNA). This diploma work will combine and modify these two ideas to fabricate a improved DNA-chip.</p><p>Immobilizing of DNA onto surface has been done by using soft lithography. Hydrophobic pattern arises from the poly(dimethylsiloxane) (PDMS) stamp. The hydrophobic pattern will attract chol-DNA that is adsorbed to the chip. Different sets of fluorophores are covalently bound to the DNA and adding CPEs to the complex will make FRET occur between CPE and bound fluorophore.</p><p>We will here show that the specificity in DNA hybridization by using PDMS patterning was high. FRET clearly occurred, especially with the CPEs as donor to the fluorophore Cy5. The intensity of FRET was higher when the fluorophore and the CPE were conjugated to the same DNA strand. The largest difference in FRET intensity between double stranded and single stranded complexes was observed with the CPE tPOMT. Super FRET has been observed but not yet fully proved. The FRET efficiency was lower with the fluorophore Alexa350 as donor compared to the Cy5/CPE complex. Most of the energy transferred from Alexa350 was extinguished by quenching.</p>

La fluorescence d'assemblées de dérivés de Bodipy a été étudiée afin de réaliser des nanocapteurs. Les nanoobjets envisagés sont soit des nanocristaux soit des particules de polystyrène en suspension dans l'eau, i.e. des nanolatex. Dans un premier temps, des molécules encombrées ont été synthétisées afin de limiter les interactions entre fluorophores à l'état solide. Les résultats obtenus à l'état amorphe montrent que cette stratégie est prometteuse. Les propriétés de fluorescence de cryptobodipy ont aussi été étudiées. L'étude des structures cristallographiques de certaines de ces molécules a permis de mieux comprendre la fluorescence à l'état solide des Bodipy. Enfin, pour l'iodophénylbodipy, la formation d'agrégats de type J dans le solide a été démontrée et il est possible de préparer des nanoparticules fluorescentes à partir de cette molécule. La fluorescence de certains Bodipy en matrice polymère a aussi été étudiée. La fluorescence de films de PMMA contenant du PM597 a pu être modulée par transfert d'¤énergie vers une molécule photochrome. D'¤autre part, la fluorescence de nanolatex contenant du trimésitylbodipy à été éteinte de manière très efficace par du bleu de cibacron, ce qui montre l'intérêt de développer des nanocapteurs à partir de ces particules.

La fluorescence d'assemblées de dérivés de Bodipy a été étudiée afin de réaliser des nanocapteurs. <br />Les nanoobjets envisagés sont soit des nanocristaux soit des particules de polystyrène en suspension dans l'eau, i.e. des nanolatex.<br /><br />Dans un premier temps, des molécules encombrées ont été synthétisées afin de limiter les interactions entre fluorophores à l'état solide. Les résultats obtenus à l'état amorphe<br />montrent que cette stratégie est prometteuse. Les propriétés de fluorescence de cryptobodipy ont aussi été étudiées. L'étude des structures cristallographiques de certaines de ces <br />molécules a permis de mieux comprendre la fluorescence à l'état solide des Bodipy. Enfin, pour l'iodophénylbodipy, la formation d'agrégats de type J dans le solide a <br />été démontrée et il est possible de préparer des nanoparticules fluorescentes à partir de cette molécule.<br /><br />La fluorescence de certains Bodipy en matrice polymère a aussi été étudiée. La fluorescence de films de PMMA contenant du PM597 a pu être modulée par transfert d'énergie<br /> vers une molécule photochrome. D'autre part, la fluorescence de nanolatex contenant du trimésitylbodipy a été éteinte de manière très efficace par du bleu de cibacron, ce qui montre <br /> l'intérêt de développer des nanocapteurs à partir de ces particules.

Different optical and calorimetric techniques were utilized to gain a better understanding of aggregated macromolecules. This research looked at two different macromolecules: poly(9,9'-dioctylfluorene), a conjugated polymer that forms aggregates in organic solvents; and bovine insulin, which forms amyloid fibrils. Conjugated polymers are of increasing interest due to their thermal stability and ease of solution processing for use in devices. A member of the polyfluorene family, poly(9,9'-dioctylfluorene) (PFO), has been studied due to its blue-emitting spectral properties. However, PFO has been found to form aggregates in solution, which is detected by the presence of a red-shifted absorption peak. This peak is caused when a section of the backbone planarizes forming the [beta]-phase. The [beta]-phase can be removed from the solution upon heating and will not return until the solution is cooled, making it a non-equilibrium process. The dissolution and reformation of the -phase were monitored using absorption spectroscopy and differential scanning calorimetry. Atomic force microscopy (AFM) and near-field scanning optical microscopy (NSOM) were able to probe the aggregates in films. It is important to understand polymer properties in solution in order to understand film morphology. Amyloid fibrils contribute to over 20 different neurodegenerative diseases, in which cures have yet to be found. The fibrils form when a soluble protein misfolds and self-assembles to form insoluble protein aggregates, and the cause of the fibril formation in vivo has still yet to be determined. Spectroscopy studies have been made possible with the use of fluorescent dyes: thioflavin T (ThT), BTA-2, and Congo red (CR). These dyes bind to amyloid fibrils and exhibit changes in their spectral properties. However, the exact mechanism for the binding of these dyes has only recently been studied. Through the use of calorimetry, the forces involved with binding of ThT and CR to amyloid fibrils can be determined. Absorption and fluorescence spectroscopy techniques were employed to study the spectral properties of these dyes. Polarized NSOM was used to determine the ThT or BTA-2's orientation with an individual fibril. Understanding how these dyes bind to fibrils will enable researchers to use spectroscopy to study the early stages of fibril formation. / text

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) undergoes homotypic and heterotypic cis- and trans- interactions that regulate processes including metabolism, immune response, and tumorigenesis. To better understand and eventually control CEACAM1’s numerous roles, we characterized the localization, homotypic cis-oligomerization, and regulation of CEACAM1 at the molecular scale using steady-state TIRFPM homo-FRET imaging in living cells. We established the anisotropy sensitivity of our TIRFPM platform using Venus monomers and dimers, which had significantly different anisotropy values. Heterogeneously distributed across the plasma membrane, CEACAM1-4L-EYFP was a mixture of monomers and oligomers, with a slightly more monomeric population at the high intensity regions. In addition, perturbation with ionomycin or α-CEA pAb increased CEACAM1 monomers, potentially in a localized manner. Although limited in detecting any anisotropy differences between CEACAM1-4L-EYFP and monomeric G432,436L-CEACAM1-4L-EYFP populations, TIRFPM homo-FRET imaging can be a useful tool for studying membrane protein self-association with proper controls and studies that focus on relative anisotropy changes.

The lower oxygen limit (LOL) in plants marks the oxygen (O2) level where the metabolism shifts from being predominantly aerobic to anaerobic; recent work has shown that respiratory-based indicators of this metabolic shift are well-correlated with changes in chlorophyll a fluorescence signals. The physiological and biochemical changes at the root of this relationship have not been well-described in the literature. The processes involved are spatially separated: chlorophyll fluorescence is associated with the lightdependent reactions and emanates from the chloroplasts whereas aerobic respiration and fermentation occurs in the mitochondria and cytosol, respectively. Evidences outlined in this thesis are used to suggest the mechanistic link between these three regions of the cell is a fluid exchange of cellular reductant. When mitochondrial respiration is inhibited as a result of inadequate O2, used as a terminal electron acceptor, glycolytic reductant in the form of NADH accumulates in the cytosol. Reductant imbalances between the cytosol and organelles can be adjusted indirectly using translocators. Excess chloroplastic reductant is used to reduce the plastoquinone (PQ) pool via NADPH-dehydrogenase, a component of the chlororespiratory pathway, effectively decreasing the photochemical quenching (qP) capacity thereby inducing a switch from minimum fluorescence (Fo) to a higher relative fluorescence (F) value where qP < 1. Subjecting dark-adapted photosystems to low-intensity light increased Fo to a slightly higher F value due to a lightinduced reduction of the oxidized PQ pool when the O2 was above the LOL, but decreased F as a result of a PSI-driven oxidation of the already over-reduced PQ pool when the O2 was below the LOL. Low O2 was also shown to increase violaxanthin deepoxidation and non-photochemical quenching (qN), likely a reflection of the overreduced state of the photosystems and associated pH decrease. Dynamic controlled atmosphere (DCA) is a fluorescence-based controlled atmosphere (CA) system that sets the optimum atmosphere for fruits and vegetables based on a product’s fluorescence response. Experiments in this thesis on the relationship between O2, temperature, light, metabolism, pigmentation and chlorophyll fluorescence were used to interpret the physiology behind fluorescence changes, suggest improved DCA techniques and outline potentially profitable avenues for future research.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) undergoes homotypic and heterotypic cis- and trans- interactions that regulate processes including metabolism, immune response, and tumorigenesis. To better understand and eventually control CEACAM1’s numerous roles, we characterized the localization, homotypic cis-oligomerization, and regulation of CEACAM1 at the molecular scale using steady-state TIRFPM homo-FRET imaging in living cells. We established the anisotropy sensitivity of our TIRFPM platform using Venus monomers and dimers, which had significantly different anisotropy values. Heterogeneously distributed across the plasma membrane, CEACAM1-4L-EYFP was a mixture of monomers and oligomers, with a slightly more monomeric population at the high intensity regions. In addition, perturbation with ionomycin or α-CEA pAb increased CEACAM1 monomers, potentially in a localized manner. Although limited in detecting any anisotropy differences between CEACAM1-4L-EYFP and monomeric G432,436L-CEACAM1-4L-EYFP populations, TIRFPM homo-FRET imaging can be a useful tool for studying membrane protein self-association with proper controls and studies that focus on relative anisotropy changes.

Methods were developed to study delayed light emission (luminiscence) and fluorescence changes in intact leaves of plants. Delayed light emission, detected from plants in darkness, was used to produce images of the plant leaves. The procedure was termed "phytoluminography". The use of the method is suggested for dia- nostic purposes at early stages of disturbances of the leaf tissues, not detectable with the naked eye. The delayed light emission is associated with the photochemistry of photosystem II and the light induced conversion and storage of energy in the thylakoid membrane system of chloroplasts. Fluorescence yield changes were induced by lowering temperature between 20 C and -20 C. The temperature induced fluorescence changes in leaves parallel the temperature induced changes in isolated chloroplasts in reaction preparations mediating photosynthetic electron transport from endogenous water splitting to added NADP. At above freezing temperatures, lowering the temperature at a constant rate of 1 C per minute caused supressed electron transport and increased fluorescence yield which were linearely dependent on the temperature change in frost resistent plants. Repeated freeze-thaw cycles between 20 °C and -20 °C induced variable fluorescence yield changes which were gradually depleated to F0 or Fm when the electron transport was injuried on the oxidizing or on the reduzing side of photosystem II, respectively. The temperature induced fluorescence changes were used to characterize plants with different ability to withstand freezing temperatures. The method also discriminates between plants of different frost resistance, and the method was used in screening for frost tolerance. / <p>Diss. (sammanfattning) Umeå : Umeå universitet, 1981, härtill 5 uppsatser.</p> / digitalisering@umu