<p>A gill filament-based assay for the cytochrome P450 1A (CYP1A)-catalysed activity ethoxyresorufin <i>O</i>-deethylase (EROD) was developed in rainbow trout (<i>Oncorhynchus mykiss</i>) and applied to Atlantic salmon (<i>Salmo salar</i>), Arctic charr (<i>Salvelinus alpinus</i>), Atlantic cod (<i>Gadus morhua</i>), saithe (<i>Pollachius virens</i>), and spotted wolffish (<i>Anarhichas minor</i>). Exposure to waterborne β-naphthoflavone (βNF; 10^-6 M) induced branchial EROD activity in all species but the spotted wolffish. In rainbow trout exposed to low concentrations of benzo[a]pyrene (BaP; 10^-9 M) and the textile dye indigo (10^-8 M) the gills responded more rapidly than the liver to BaP, and indigo induced branchial but not hepatic EROD activity.</p><p>A CYP1A-dependent BaP adduct formation was shown in gills of fish exposed to waterborne ³H-BaP, i.e. the adduct formation was enhanced by βNF and blocked by ellipticine (CYP1A inhibitor). The predominant location for BaP adducts was the secondary lamellae (most exposed part of the gill filament), whereas the CYP1A enzyme was also present in the primary lamellae of the gill filament. Hence, in addition to the cell-specific expression of CYP1A an important determinant for the localisation of adducts seemed to be the bioavailability of BaP. This idea is supported by the fact that the CYP1A enzyme was induced only in secondary lamellae by BaP (10^-7 M) and indigo (10^-6 M), whereas it was induced in both primary and secondary lamellae by 3,3´,4,4´,5-pentachlorobiphenyl (10^-8 M). Apparently, readily metabolised inducers (BaP and indigo) are biotransformed in the secondary lamellae.</p><p>My results show that gill filament EROD activity is a sensitive biomarker of exposure to waterborne dioxin-like pollutants, and that the assay has potential for use in monitoring. Furthermore, the results suggest that readily metabolised dioxin-like compounds absorbed via the gills may undergo first-pass metabolism in the gill cells and therefore remain undetected by monitoring of EROD activity in the liver.</p>

A gill filament-based assay for the cytochrome P450 1A (CYP1A)-catalysed activity ethoxyresorufin O-deethylase (EROD) was developed in rainbow trout (Oncorhynchus mykiss) and applied to Atlantic salmon (Salmo salar), Arctic charr (Salvelinus alpinus), Atlantic cod (Gadus morhua), saithe (Pollachius virens), and spotted wolffish (Anarhichas minor). Exposure to waterborne β-naphthoflavone (βNF; 10-6 M) induced branchial EROD activity in all species but the spotted wolffish. In rainbow trout exposed to low concentrations of benzo[a]pyrene (BaP; 10-9 M) and the textile dye indigo (10-8 M) the gills responded more rapidly than the liver to BaP, and indigo induced branchial but not hepatic EROD activity. A CYP1A-dependent BaP adduct formation was shown in gills of fish exposed to waterborne 3H-BaP, i.e. the adduct formation was enhanced by βNF and blocked by ellipticine (CYP1A inhibitor). The predominant location for BaP adducts was the secondary lamellae (most exposed part of the gill filament), whereas the CYP1A enzyme was also present in the primary lamellae of the gill filament. Hence, in addition to the cell-specific expression of CYP1A an important determinant for the localisation of adducts seemed to be the bioavailability of BaP. This idea is supported by the fact that the CYP1A enzyme was induced only in secondary lamellae by BaP (10-7 M) and indigo (10-6 M), whereas it was induced in both primary and secondary lamellae by 3,3´,4,4´,5-pentachlorobiphenyl (10-8 M). Apparently, readily metabolised inducers (BaP and indigo) are biotransformed in the secondary lamellae. My results show that gill filament EROD activity is a sensitive biomarker of exposure to waterborne dioxin-like pollutants, and that the assay has potential for use in monitoring. Furthermore, the results suggest that readily metabolised dioxin-like compounds absorbed via the gills may undergo first-pass metabolism in the gill cells and therefore remain undetected by monitoring of EROD activity in the liver.

The biological function of a protein is determined by its amino acid sequence, structure, and internal dynamics. In turn the prediction of a protein structure from its folding pathway involves the characterization of the dynamics of the polypeptide backbone. This study addresses how the internal dynamics of arborescent polypeptides are affected by increased crowding of the interior of these branched polymer molecules. Linear, comb-branched, and arborescent poly(L-glutamic acid) (PGA) samples were analyzed by 1H NMR spectroscopy to determine their chain conformation. The PGA chains of these constructs were shown to adopt α-helical and random coil conformations in N,N-dimethylformamide (DMF) and in dimethyl sulfoxide (DMSO), respectively. The hydrodynamic diameter (Dh) of the arborescent PGAs, determined using dynamic light scattering measurements, increased with increasing generation number and when the side-chains adopted random coil instead of α-helical conformations. The PGA samples were labelled with 1-pyrenemethylamine to determine how their structure affected the internal dynamics of the arborescent polymers in solution, from the analysis of their fluorescence spectra and decays. For each pyrene-labelled polymeric construct excimer formation increased with increasing pyrene content, and the efficiency of excimer formation increased with the generation number due to the increased density of the macromolecules. Comparison of the time-resolved fluorescence results acquired in DMF and in DMSO demonstrated that the helical conformation led to slower chain dynamics in DMF and that despite the higher viscosity of DMSO, the polypeptide side-chains were more mobile as a consequence of the random coil conformation of the linear PGA segments. These results suggest that the formation of structural motives inside a polypeptide slows down its internal dynamics.

Smoking and environmental tobacco smoke (ETS) exposure are major risk factors for cardiovascular disease (CVD), although the exact components and pathophysiological mechanisms responsible for this association remain unclear. Polycyclic aromatic hydrocarbons (PAHs), including benzo[a]pyrene (BaP), are ubiquitous environmental contaminants that form during organic material combustion and are thus found in cigarette smoke, vehicle exhaust particles, and air pollution. We hypothesize that PAHs are key agents responsible for mediating the cigarette smoke effects in the cardiovascular system, including increased oxidative stress, inflammation, and arterial stiffness.<p> Arterial stiffness is a powerful, independent predictor of cardiovascular risk and is regulated, in part, by vasoactive mediators derived from the endothelium. The first objective of this project was to determine whether pulse wave dP/dt collected from radiotelemetry-implanted rats is a reliable indicator of changes in arterial stiffness following administration of vasoactive drugs or acute ETS exposure. Anaesthetized rats were administered a single dose of saline (vehicle control), acetylcholine, norepinephrine, and N(G)-nitro-L-arginine methyl ester (L-NAME) via the tail vein, allowing a washout period between injections. Acetylcholine decreased and norepinephrine increased dP/dt compared to saline vehicle. Injection of the nitric oxide (NO) synthase inhibitor L-NAME decreased plasma nitrate/nitrite (NOx), but transiently increased dP/dt. For the ETS experiment, rats were exposed for one hour to sham, low dose ETS, or high dose ETS. Exposure to ETS did not significantly alter dP/dt or plasma endothelin-1 (ET-1) levels, but increased plasma NOx levels at the high ETS exposure and increased plasma nitrotyrosine levels in both ETS groups. In conclusion, acute changes in NO production via acetylcholine or L-NAME alter the arterial pulse wave dP/dt consistently with the predicted changes in arterial stiffness. Although acute ETS appears to biologically inactivate NO, a concomitant increase in NO production at the high ETS exposure may explain why ETS did not acutely alter dP/dt.<p> The second objective of this project was to compare the effects of subchronic ETS and BaP exposure on circadian blood pressure patterns, arterial stiffness, and possible sources of oxidative stress in radiotelemetry-implanted rats. Pulse wave dP/dt was used as an indicator of arterial stiffness, and was compared to both structural (wall thickness) and functional (NO production and bioactivity, ET-1 levels) features of the arterial wall. In addition, histology of lung, heart, and liver were examined as well as pulmonary and hepatic detoxifying enzyme activity (cytochrome P450 specifically CYP1A1). Daily ETS exposure for 28 days altered the circadian pattern of heart rate and blood pressure in rats, with a loss in the normal dipping pattern of blood pressure during sleep. Subchronic ETS exposure also increased dP/dt in the absence of any structural modifications in the arterial wall. Although NO production and ET-1 levels were not altered by ETS, there was increased biological inactivation of NO via peroxynitrite production (as indicated by increased plasma nitrotyrosine levels). Thus, vascular stiffness and failure of blood pressure to dip precede structural changes in rats exposed to ETS for 28 days. Exposure to ETS also caused increased number of lung neutrophils as well as increased CYP1A1 activity in lung microsomes.<p> Since ETS-induced increases in arterial stiffness occurred as early as day 7, radiotelemetry-implanted rats were exposed daily to intranasal BaP for 7 days. Similar to ETS, BaP exposure altered circadian blood pressure patterns and reduced blood pressure dipping during sleep. Thus, in support of part of our hypothesis, the PAH component of cigarette smoke may be responsible for the ETS-induced increase in blood pressure and the loss of dipping pattern during sleep. Increased neutrophil recruitment was observed in the lungs of both ETS- and BaP-exposed rats, suggesting that lung inflammatory reactions may be involved in the disruption of circadian blood pressure rhythms. Unlike ETS however, BaP exposure did not significantly alter pulse wave dP/dt, endothelial function, or lung CYP1A1 activity. Thus, contrary to our hypothesis, the reduction in NO bioactivity and increased arterial stiffness caused by ETS cannot be explained by BaP at the dose and length of the exposure in the current study. Production of reactive metabolites in the lung following ETS exposure may be responsible, at least in part, for the increases in oxidative stress in the vasculature, leading to reduced NO bioactivity and increased arterial stiffness. Oxidative stress caused by BaP exposure may have been insufficient to reduce NO bioactivity in the peripheral vasculature. Therefore arterial stiffness was not increased and factors other than NO may be responsible for the increase in blood pressure observed with ETS and BaP exposure.

Time-resolved fluorescence was applied to linear and branched pyrene-labelled macromolecules to study their internal dynamics. The linear macromolecules consisted of two series of pyrene-labelled poly(N-isopropylacrylamide)s where the polymer was either end-labelled (Py2-PNIPAM-Y where Y represents the molecular weight of the polymer and equals 6, 8, 14, 25, and 45 kDa) or randomly labelled (Py-PNIPAM-X% where X represents the pyrene content and is equal to 0.1, 2, 3, 4, 5, and 6 mol%) with pyrene. Four dendrimer generations based on a bis(hydroxymethyl)propionic acid backbone represented the branched macromolecules where the terminal sites were labelled with pyrene (PyX-GY-COOH where X represents the number of pyrene units incorporated into the Y`th generation dendrimer). A polystyrene-dendrimer hybrid was also synthesized (PyX-GY-PS). The fluorescence decays of the Py2-PNIPAM-Y and Py-PNIPAM-X% samples were acquired in solvents of varying viscosity and were analyzed with the Birks Scheme and the Fluorescence Blob Model (FBM) to yield the excimer formation rate constants and , respectively. The two parameters showed the same trends with varying viscosity, implying that the same information concerning chain dynamics is obtained from the randomly and end-labelled PNIPAM samples. The fluorescence decays of the Py2-PNIPAM-Y samples were acquired in ethanol and in water to determine how pyrene solubility affects the behavior of the polymers in solution, as probed by time-resolved fluorescence. It was found that the decreased pyrene solubility in water led to large amounts of intra- and intermolecular pyrene aggregation. Finally, the pyrene-labelled dendrimers were studied in tetrahydrofuran (THF) to probe the mobility of the chain ends as a function of generation number. The average rate of excimer formation, , obtained from the Model-Free analysis of the fluorescence decays in THF, increased linearly with generation number. This finding, combined with molecular mechanics optimizations, led to the conclusion that excimer formation was greatly enhanced due to the branched nature of the dendrimer molecule. Together, these studies illustrate three different applications of the use of time-resolved fluorescence to characterize the internal dynamics of pyrene-labelled macromolecules.

A series of pyrene end-labeled monodisperse poly(ethylene oxide)s (PEO(X)-Py2 where X represents the number average molecular weight (Mn) of the PEOs and equals 2, 5, 10 and 16.5 K) and one pyrene mono-labeled PEO (PEO(2K)-Py1) were synthesized and characterized in solution using fluorescence. First, the end-to-end cyclization (EEC) of PEO(X)-Py2 was investigated in seven organic solvents with viscosities (η) ranging from 0.32 to 1.92 mPa•s. The classical Birks scheme was used to globally fit the pyrene monomer and excimer fluorescence decays. The fraction of pyrenes that did not form excimer (ffree) was found to increase with increasing η and Mn. This result was contrary to the assumptions made by Birks’ scheme. To account for this, ffree was assumed to represent the fraction of PEO chains other than the monolabeled polymer impurities that cannot accomplish EEC. A fluorescence blob model (FBM) was applied to handle this assumption in the process of excimer formation for the PEO(X)-Py2 samples in solution. The radius of a blob, Rblob, in organic solvents was determined according to the results retrieved from the FBM. To quantitatively account for the existence of pyrene impurity in pyrene-labeled macromolecules, known amounts of PEO(2K)-Py1 were added into a PEO(2K)-Py2 solution and the fluorescence decays were fitted globally according to the Birks scheme and “model free” (MF) analysis to verify the validation of the MF analysis. The MF analysis was then applied to determine the amounts of 1-pyrenebutyric acid (PyBA) that had been added to a solution of pyrene end-labeled fourth generation dendritic hybrid (Py16-G4-PS). The results demonstrated that the contribution from unwanted fluorescent species could be isolated and quantitatively accounted for by fitting the fluorescence decays of the pyrene monomer and excimer globally with the MF analysis. Since the PEO(X)-Py2 samples form hydrophobic pyrene aggregates in aqueous solution, a sequential model (SM) was proposed to characterize the pyrene excimer formation of PEO(X)-Py2 in water at different polymer concentration (CP). The capture distance over which the pyrenyl end-groups experience hydrophobic forces in water was determined by assuming that the end-to-end distances of the PEO(X)-Py2 samples adopt a Gaussian distribution and that the fraction of pyrenes that are aggregated (fE0) determined by the sequential model corresponds to the fraction of PEO(X)-Py2 chains whose end-to-end distance is smaller than the hydrophobic capture distance. Since a surfactant can interact with a hydrophobically modified water-soluble polymer in aqueous solution, the interactions taking place between PEO(X)-Py2 and sodium dodecyl sulfate (SDS) were investigated at a low PEO(X)-Py2 concentration. The pyrene monomer and excimer fluorescence decays of the PEO(X)-Py2 and SDS solutions were acquired at various SDS concentrations and globally fitted according to the MF analysis to retrieve the parameters that described the kinetics of pyrene excimer formation. At high SDS concentrations above the critical micelle concentration (CMC), the pyrene end-groups of the short-chain samples (PEO(2K)-Py2 and PEO(5K)-Py2) were incorporated inside the same micelle and excimer was formed intramolecularly, while most pyrene groups of the long-chain samples (PEO(10K)-Py2 and PEO(16.5K)-Py2) were isolated into different micelles. Lastly, both the rheological properties and fluorescence behavior of a pyrene-labeled hydrophobically-modified alkali-swellable emulsion (Py-HASE) polymer in basic aqueous solution with SDS were studied. Furthermore, a joint experimental setup that combined a rheometer and a steady-state fluorometer was applied to investigate at the molecular level the effect that a shearing force had on the polymeric network. However, despite the dramatic decrease in solution viscosity with increasing shear rate, no change in the fluorescence spectra was detected, suggesting that changes in the polymeric network that affected the balance of intra- versus intermolecular pyrene associations did not impact the process of excimer formation. Together the experiments described in this thesis represent the broadest set of examples found in the scientific literature where information on the dynamics and level of association of pyrene-labeled polymers has been retrieved through the quantitative analysis of the fluorescence decays acquired with pyrene-labeled polymers in solution.

Smoking and environmental tobacco smoke (ETS) exposure are major risk factors for cardiovascular disease (CVD), although the exact components and pathophysiological mechanisms responsible for this association remain unclear. Polycyclic aromatic hydrocarbons (PAHs), including benzo[a]pyrene (BaP), are ubiquitous environmental contaminants that form during organic material combustion and are thus found in cigarette smoke, vehicle exhaust particles, and air pollution. We hypothesize that PAHs are key agents responsible for mediating the cigarette smoke effects in the cardiovascular system, including increased oxidative stress, inflammation, and arterial stiffness.<p> Arterial stiffness is a powerful, independent predictor of cardiovascular risk and is regulated, in part, by vasoactive mediators derived from the endothelium. The first objective of this project was to determine whether pulse wave dP/dt collected from radiotelemetry-implanted rats is a reliable indicator of changes in arterial stiffness following administration of vasoactive drugs or acute ETS exposure. Anaesthetized rats were administered a single dose of saline (vehicle control), acetylcholine, norepinephrine, and N(G)-nitro-L-arginine methyl ester (L-NAME) via the tail vein, allowing a washout period between injections. Acetylcholine decreased and norepinephrine increased dP/dt compared to saline vehicle. Injection of the nitric oxide (NO) synthase inhibitor L-NAME decreased plasma nitrate/nitrite (NOx), but transiently increased dP/dt. For the ETS experiment, rats were exposed for one hour to sham, low dose ETS, or high dose ETS. Exposure to ETS did not significantly alter dP/dt or plasma endothelin-1 (ET-1) levels, but increased plasma NOx levels at the high ETS exposure and increased plasma nitrotyrosine levels in both ETS groups. In conclusion, acute changes in NO production via acetylcholine or L-NAME alter the arterial pulse wave dP/dt consistently with the predicted changes in arterial stiffness. Although acute ETS appears to biologically inactivate NO, a concomitant increase in NO production at the high ETS exposure may explain why ETS did not acutely alter dP/dt.<p> The second objective of this project was to compare the effects of subchronic ETS and BaP exposure on circadian blood pressure patterns, arterial stiffness, and possible sources of oxidative stress in radiotelemetry-implanted rats. Pulse wave dP/dt was used as an indicator of arterial stiffness, and was compared to both structural (wall thickness) and functional (NO production and bioactivity, ET-1 levels) features of the arterial wall. In addition, histology of lung, heart, and liver were examined as well as pulmonary and hepatic detoxifying enzyme activity (cytochrome P450 specifically CYP1A1). Daily ETS exposure for 28 days altered the circadian pattern of heart rate and blood pressure in rats, with a loss in the normal dipping pattern of blood pressure during sleep. Subchronic ETS exposure also increased dP/dt in the absence of any structural modifications in the arterial wall. Although NO production and ET-1 levels were not altered by ETS, there was increased biological inactivation of NO via peroxynitrite production (as indicated by increased plasma nitrotyrosine levels). Thus, vascular stiffness and failure of blood pressure to dip precede structural changes in rats exposed to ETS for 28 days. Exposure to ETS also caused increased number of lung neutrophils as well as increased CYP1A1 activity in lung microsomes.<p> Since ETS-induced increases in arterial stiffness occurred as early as day 7, radiotelemetry-implanted rats were exposed daily to intranasal BaP for 7 days. Similar to ETS, BaP exposure altered circadian blood pressure patterns and reduced blood pressure dipping during sleep. Thus, in support of part of our hypothesis, the PAH component of cigarette smoke may be responsible for the ETS-induced increase in blood pressure and the loss of dipping pattern during sleep. Increased neutrophil recruitment was observed in the lungs of both ETS- and BaP-exposed rats, suggesting that lung inflammatory reactions may be involved in the disruption of circadian blood pressure rhythms. Unlike ETS however, BaP exposure did not significantly alter pulse wave dP/dt, endothelial function, or lung CYP1A1 activity. Thus, contrary to our hypothesis, the reduction in NO bioactivity and increased arterial stiffness caused by ETS cannot be explained by BaP at the dose and length of the exposure in the current study. Production of reactive metabolites in the lung following ETS exposure may be responsible, at least in part, for the increases in oxidative stress in the vasculature, leading to reduced NO bioactivity and increased arterial stiffness. Oxidative stress caused by BaP exposure may have been insufficient to reduce NO bioactivity in the peripheral vasculature. Therefore arterial stiffness was not increased and factors other than NO may be responsible for the increase in blood pressure observed with ETS and BaP exposure.

Fluorescent labeling can improve the detection sensitivity in capillary electrophoretic (CE) separations down to attomolar concentrations. However, most fluorescent labels are not compatible with CE because their fluorescence properties and charge states are pH-dependent, they are often hydrophobic and they have a tendency to significantly change the properties of the analytes after labeling. A group of fluorescent labeling reagents have been prepared whose fluorophores have properties that are optimized for CE separations. These fluorophores have fluorescence properties and charge states that are independent of pH in the 2 < pH < 11 range. Their excitation maxima are also compatible with the 488 nm line of the Argon ion laser. A mono-cationic acridine-based fluorescent label was prepared and was found to not shift the pI of a labeled model protein in capillary isoelectric focusing separation (cIEF). Lower loading, due to increased sensitivity, led to better resolution of closely spaced isoform peaks having a pI = 0.05. A tri-anionic pyrene-based fluorescent labeling reagent was also synthesized and was used in the sodium dodecyl sulfate capillary gel electrophoresis (SDS-CGE) separation of proteins. The fluorophore led to an LOQ in the nM range, and did not alter the migration behavior of proteins in the sieving matrix. A third fluorescent labeling reagent was developed as a solid phase reagent (SPR) where the fluorophore was immobilized on a solid surface through a cleavable anchor. The fluorophore is di-anionic and is based on pyrene. The SPR was designed to allow the simultaneous capture and labeling of an analyte and the efficient release of the label-analyte conjugate under mild acidic conditions. The use of the SPR allowed the labeling of a diamine whose concentration was in the low nanomolar range. The SPR opens up the possibility for mono-labeling and proportional multiple labeling of proteins.

The purpose of this study was to look at the genotoxic and cytotoxic effects of benzo-á-pyrene (BáP), a chemical mutagen that is present in cigarette smoke, on the insulin-like growth factor-I (IGF-I) gene. Women who smoke during pregnancy are more likely to have a growth-restricted baby. We hypothesized that BáP exerts its effects through genotoxic and cytotoxic avenues. The cytotoxicity is manifested by chromosomal abnormalities and a decrease in the rate of cell division. The genotoxicity is manifested by changes in certain genes known to be important in mammalian fetal development such as IGF-I. IGF-I is implicated in intrauterine growth restriction (IUGR), a problem that greatly increases the risk of perinatal morbidity and mortality. To futher understand the mechanism by which BáP influences the normal growth and development of human placental cells, human placental trophoblast cells from an established immortalized cell line were utilized. Cells were cultured in appropriate media, starved (using starvation "Serum Free Medium"), and treated with two doses of BáP, 1µM (dose 1) and 5µM (dose 2). Chromosomes were prepared for cytogenetic analysis and visualized using light microscopy after Giemsa staining. Chromosomal aberrations were identified and the rate of cell division was determined through the analysis of the mitotic index for treated cells compared to a control group. To further understand the influence of BáP on the IGF-I gene expression level, RNA was extracted from control and treated cells, from which cDNA was synthesized and used for further analysis using polymerized chain reaction (PCR). The PCR results were used to better understand the genotoxicity of BáP, while chromosomal aberration analysis was used to determine the cytotoxic effects of BáP on human placental cells. Our results indicate that many chromosomal abnormalities were present in the treated groups compared to the control group. In addition, there was a significant decrease in the mitotic index of the BáP-treated cells (MI=0.3%) verses the control group (MI=0.93%), p value 0.0447. Through the PCR assay, we speculate that there is a dose-related response to BáP of the IGF-I RNA expression level, with low levels in the treated groups compared to the control group. We conclude from these results that BáP influences placental cells at both the gene and chromosome level. It also affects the cell cycle of human placental cells. It is known that smoking is deleterious for fetal development. We believe that the current study brings us closer to understanding the mechanism by which smoking can lead to fetal growth restriction.

Time-resolved fluorescence was applied to linear and branched pyrene-labelled macromolecules to study their internal dynamics. The linear macromolecules consisted of two series of pyrene-labelled poly(N-isopropylacrylamide)s where the polymer was either end-labelled (Py2-PNIPAM-Y where Y represents the molecular weight of the polymer and equals 6, 8, 14, 25, and 45 kDa) or randomly labelled (Py-PNIPAM-X% where X represents the pyrene content and is equal to 0.1, 2, 3, 4, 5, and 6 mol%) with pyrene. Four dendrimer generations based on a bis(hydroxymethyl)propionic acid backbone represented the branched macromolecules where the terminal sites were labelled with pyrene (PyX-GY-COOH where X represents the number of pyrene units incorporated into the Y`th generation dendrimer). A polystyrene-dendrimer hybrid was also synthesized (PyX-GY-PS). The fluorescence decays of the Py2-PNIPAM-Y and Py-PNIPAM-X% samples were acquired in solvents of varying viscosity and were analyzed with the Birks Scheme and the Fluorescence Blob Model (FBM) to yield the excimer formation rate constants and , respectively. The two parameters showed the same trends with varying viscosity, implying that the same information concerning chain dynamics is obtained from the randomly and end-labelled PNIPAM samples. The fluorescence decays of the Py2-PNIPAM-Y samples were acquired in ethanol and in water to determine how pyrene solubility affects the behavior of the polymers in solution, as probed by time-resolved fluorescence. It was found that the decreased pyrene solubility in water led to large amounts of intra- and intermolecular pyrene aggregation. Finally, the pyrene-labelled dendrimers were studied in tetrahydrofuran (THF) to probe the mobility of the chain ends as a function of generation number. The average rate of excimer formation, , obtained from the Model-Free analysis of the fluorescence decays in THF, increased linearly with generation number. This finding, combined with molecular mechanics optimizations, led to the conclusion that excimer formation was greatly enhanced due to the branched nature of the dendrimer molecule. Together, these studies illustrate three different applications of the use of time-resolved fluorescence to characterize the internal dynamics of pyrene-labelled macromolecules.