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Voltage clamp fluorometry (VCF) was first developed in the mid 1990s by Isacoff and his colleagues. In this approach fluorophores are attached to substituted cysteine residues that are engineered by site-directed mutagenesis. Changes in the dielectric environment of the fluorophore report local transitions that are associated with electrically-related and electrically-silent transitions. VCF provides a powerful technique to observe real time reports of ion channel gating conformations. It has proven to be a useful technique because it adds insight that is not available using other techniques. X-ray crystallography studies give a predominantly static picture of the channel, while patch clamping of channels gives information only about residues that effect ionic current flow. Similarly, gating current provides insight only about residues that are charged and move across the membrane electric field. In this thesis we examined the structural rearrangements of the Shaker channel and the effect of 4-AP on channel gating. We also examined for the first time the structural rearrangements of the Kv1.5 gating and the how the channel responds to depolarization pulses. This work is instrumental in the examination of the potassium channel gating.

Antithrombin (AT) is one of the most important anticoagulant factors in the blood, and its effects are increased by the interaction with glycosaminoglycans, especially heparin. AT appears in two additional variants, other than the native form, and those variants have antiangiogenic properties and also bind to heparin. AT is found in two distinct isoforms (alfa, beta) where the difference lie in the degree of glycosylation. This project has shown interesting results regarding the dependence of calcium ions on the binding between heparin and antithrombin. The results show that the beta-isoform increases its affinity for heparin in the presence of calcium in contrast to the alfa-isoform, which shows a decrease in the heparin affinity under the same conditions. This project has also given results that after further investigation and development could be used for an improved set-up of the immobilisation of AT variants in a surface plasmon resonance system. The results show that immobilisation of a protein in the reference channel gives a better shielding effect between the negatively charged heparin molecules and the negatively charged dextran matrix. Furthermore a more significant difference was seen between the two heparin moieties used during binding affinity studies, especially for native AT.

<p>Antithrombin (AT) is one of the most important anticoagulant factors in the blood, and its effects are increased by the interaction with glycosaminoglycans, especially heparin. AT appears in two additional variants, other than the native form, and those variants have antiangiogenic properties and also bind to heparin. AT is found in two distinct isoforms (alfa, beta) where the difference lie in the degree of glycosylation. This project has shown interesting results regarding the dependence of calcium ions on the binding between heparin and antithrombin. The results show that the beta-isoform increases its affinity for heparin in the presence of calcium in contrast to the alfa-isoform, which shows a decrease in the heparin affinity under the same conditions. This project has also given results that after further investigation and development could be used for an improved set-up of the immobilisation of AT variants in a surface plasmon resonance system. The results show that immobilisation of a protein in the reference channel gives a better shielding effect between the negatively charged heparin molecules and the negatively charged dextran matrix. Furthermore a more significant difference was seen between the two heparin moieties used during binding affinity studies, especially for native AT.</p>

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Abstract The development of commercially available assays from the measurement of enzyme activity to mass concentrations of proteins, especially the assays of cardiac troponin I and T, has been the most important innovation in the field of cardiovascular laboratory diagnostics over the decade. The availability of a simple, rapid test using whole blood to facilitate processing and to reduce the turnaround time could improve the management of patients presenting with chest pain. The aim of this study was to evaluate the analytical and clinical performance of a new time-resolved fluorometry-based immunology technology using the cardiac marker and high-sensitivity C-reactive protein assays. In addition, the use of high-sensitivity C-reactive protein assay for the investigation of patients with acute atrial fibrillation and the influence of heparin for cardiac marker assays were studied. The levels of precision attained with pooled serum and plasma samples and control materials were acceptable. The assays were found to be linear within the ranges tested. The correlation coefficient between the Innotrac Aio! 1st generation cTnI and Abbott AxSYM cTnI assays was 0.960, and the slope was 0.07. The correlations between the 2nd generation Innotrac Aio!, Access AccuTnI and Abbott AxSYM assays were good, but there were biases between the methods. The correlation coefficients between the Innotrac Aio! and Abbott AxSYM CK-MB and myoglobin assays were 0.995 and 0.971, respectively, but the Innotrac Aio! CK-MB assay yielded about 9% higher values than the Abbott assay. The correlations between Innotrac Aio! usCRP and Cobas Integra CRP latex and between Innotrac Aio! usCRP and Hitachi CRP (Latex ) HS were good. Furthermore, the sample material correlation studies showed no significant differences when the Innotrac Aio! System was used. However, the mean Abbott AxSYM CK-MB values and the cTnI values for heparin plasma samples were 17% higher and about 15% lower than for serum samples, respectively. In the investigation of CRP levels in patients with acute atrial fibrillation CRP tended to be higher in the patients with acute FA, and there was a positive correlation between the concentrations of CRP and IL-6. The results demonstrate the excellent analytical performance of the Innotrac Aio! 2nd generation cTnI, myoglobin, CK-MB and usCRP assays, and all the matrices, including serum, plasma and whole blood, are suitable sample matrices to be used with these methods without further standardization.

A toxoplasmose, protozoose disseminada de baixa morbidade, apresenta número significativo de doença ocular, congênita ou do sistema nervoso central. O diagnóstico é sorológico por diferentes testes, mas limiares baixos e variação individual levam a frequentes problemas. Novos imunoensaios fluorescentes de fase sólida (FLISA) usam a quantificação direta de anticorpos. Aqui, desenvolvemos um FLISA multiplex (FLISAm) para a detecção simultânea de anticorpos IgG e IgM contra Toxoplasma gondii. Após padronização, a eficiência do FLISAm com conjugados comerciais foi feita inicialmente de forma isolada para cada imunoglobulina em 140 amostras de soro de universitários previamente analisadas pelo ELISA IgG/IgM. FLISA IgG mostrou boa concordância (Kappa=0,7088), com sensibilidade de 83,3% e especificidade de 94,2%, enquanto FLISA IgM apresentou boa concordância (K=0,6026), menor sensibilidade de 55,5% e igual especificidade de 98,4%. Foram produzidos novos conjugados fluorescentes de maior especificidade e seu desempenho no FLISAm foi validado em 24 amostras e sua eficiência foi avaliada em 120 amostras conhecidas de soro de gestantes. FLISAm mostrou excelente concordância, tanto para a detecção de anticorpos IgG (K=0.8837, sensibilidade=100,0%, especificidade=87,5%), quanto para a detecção de anticorpos IgM (K=0,9187, sensibilidade=100%, especificidade=99,1%) com excelente reprodutibilidade. O teste desenvolvido é rápido, econômico, de fácil execução, alto rendimento e que pode ser utilizado como método de triagem de soroconversão em mulheres grávidas, útil em aplicações de grande número de amostras como o cuidado pré-natal. / Toxoplasmosis, a disseminated low morbidity protozoan disease, presented significant numbers of affected people, mainly ocular disease, fetal infections or encephalitis in immune deficient patients. Serology is the main diagnosis with commercial antibody assays, but individual variation or low thresholds cause many inconsistencies. New solid phase immunofluorescence assays (FLISA) allows direct antibody quantification in microplates. Here, we developed a multiplex FLISA (FLISAm) for simultaneous detection of IgG and IgM anti-Toxoplasma gondii antibodies. After standardization, the efficiency of this method was initially analyzed in isolated FLISA for each immunoglobulin with commercial conjugates in 140 serum samples of the students previously screened by IgG/IgM ELISA. IgG FLISA showed good concordance (Kappa=0.7088), 83,3% sensitivity and 94,2% specificity, while IgM FLISA also showed good concordance (Kappa=0,6026), lower 55,5% sensitivity and similar 98,4% specificity. New higher efficiency conjugated were prepared and tested in 120 serum samples of the pregnant woman in a same well conjunct IgG/IgM FLISAm. We also validate the FLISAm in 24 serum samples. Compared to isolated ELISA IgG/IgM, FLISAm demonstrated excellent concordance for IgG (Kappa=0.8837; sensitivity=100%; specificity=87,5%,) and IgM (Kappa=0,9187; sensitivity=100%; specificity=99,1%), with excellent reproducibility. The standardized FLISAm is quick, inexpensive, easily performed and high throughput and the assay can be used for screening serum conversion in pregnant women, useful in large numbers applications as antenatal care.

Much of the aerosol research completed at the University of South Florida has revolved around evaluating industrial hygiene equipment and instrumentation in environmental chambers. Data collected during these studies has provided valuable baseline data on equipment and instrument performance under calm air conditions. A newly constructed wind tunnel now allows researchers to evaluate industrial hygiene instruments under moving air conditions. Because the wind tunnel is capable of producing wind velocities that a worker could encounter in the occupational setting, researchers may gain insight into instrument performance under simulated field conditions. Because aerosols can be introduced into the new wind tunnel testing section, researchers can also challenge industrial hygiene equipment and instrumentation with aerosols in sizes ranges that are of interest in public health. The purpose of this dissertation research was to develop a new wind tunnel to be used for aerosol research at the University of South Florida. Three specific aims had to be met for this study to be successful. They included: (1) designing a wind tunnel based on best practice information outlined in scientific literature, (2) constructing an operable wind tunnel to be used for aerosol research, and (3) characterizing wind tunnel performance by examining the wind tunnel velocity profile, turbulence intensity, and aerosol introduction/collection. The actual wind tunnel was constructed to a length of approximately 20 feet, a height of approximately 2 feet at its tallest point, and includes an entrance filter housing, a settling chamber, a contraction, a testing section, a diffuser, an exit filter housing, a fan, and exhaust duct. All components were designed and constructed using guidelines and best practices reported in the scientific literature. Velocity profile measurements were the first way that this wind tunnel was characterized. In order to successfully obtain measurements, the wind tunnel cross section was divided into 16 equal quadrants. Five measurements were taken for each quadrant at each wind velocity. Target wind velocities for this research were 0.5 m/s, 1.0 m/s, and 2.0 m/s. Actual average wind velocities of 0.48 m/s, 1.00 m/s, and 2.04 m/s. All were within established limits reported in the scientific literature. Turbulence intensity measurements were the second way that this wind tunnel was characterized. In order to successfully obtain measurements, the wind tunnel cross section was divided into 16 equal quadrants. Five measurements were taken for each quadrant at each wind velocity. Wind tunnels are typically designed to have the lowest turbulence intensity possible, generally below 10%. The overall average turbulence intensities for this wind tunnel at wind velocities of 0.5 m/s, 1.0 m/s, and 2.0 m/s were 9%, 10%, and 8% respectively. Overall turbulence intensity measurements were at or below 10%. Isokinetic sampling was the final method used to characterize this wind tunnel by collecting and detecting aerosols traveling through the wind tunnel testing section. The wind tunnel was operated at wind velocities of 0.5 m/s, 1.0 m/s, and 2.0 m/s with isokinetic sampling flow rates of 15.4 L/min, 30.9 L/min, and 61.7 L/min respectively. Monodisperse fluorescent polystyrene latex spheres were used as the test aerosol because they are uniform in size and shape and can be detected by fluorometry. The Blaustein Multi-Jet Atomizer (BLAM) was used to generate monodisperse fluorescent polystyrene latex aerosol 0.5 µm and 2.0 µm particles from liquid suspensions. The Vilnius Aerosol Generator (VAG) was used to generate monodisperse fluorescent polystyrene latex aerosol of 6.0 µm and 12.0 µm particles from dry powders. Nitrogen gas was used for delivering test aerosols into the wind tunnel. Five experimental runs were completed for each particle size and wind velocity for a total of 60 experimental runs. Fluorescence was detected in all 60 samples with average mass concentrations ranging from 0.000050 ng/ml to 0.002703 ng/ml. Based on velocity profile measurements, turbulence intensity measurements, and isokinetic sampling, the performance of University of South Florida wind tunnel was found to be excellent, indicating that it was designed and constructed appropriately. The wind tunnel can now successfully be used by researchers interested in evaluating industrial hygiene sampling equipment with aerosols ranging from 0.5 µm to 12.0 µm in moving air with velocities ranging from 0.5 m/s to 2.0 m/s.

ABSTRACT Benthic foraminifers that host algal symbionts are similar to corals in that they rely on their algal endosymbionts for their energy needs, calcify prolifically, and are sensitive to changes in environmental conditions. They are abundant in the benthos of coastal coral-reef areas and are found throughout the tropical and subtropical regions. Pulse Amplitude Modulated (PAM) chlorophyll fluorometry and chlorophyll a extraction techniques were used to quantify and compare the photosynthetic responses of the benthic foraminiferal, Archaias angulatus and their isolated endosymbionts, Chlamydomonas hedleyi, to short-term changes in temperature. Maximum quantum efficiency (Fv/Fm) and rapid light curves (RLCs), from which relative electron transport rates (rETR) of photosystem II (PSII) were derived, were investigated over a thermal range from 4.4° to 33.9 °C in three experiments that were 7 to 31 days in duration. Typical mean yields (Fv/Fm for healthy holobionts (symbionts in hospite) were 0.6 - 0.7, and for isolated symbionts 0.5 - 0.6. Chronic photoinhibition, indicated by significant decreases in Fv/Fm, occurred at temperatures above 31.0°C; there was minimal reduction in efficiency in cooler treatments. The trends between holobiont and symbionts were very similar in all of the photophysiological parameters measured [yield, photoefficiency (<α>), ETRmax and minimum saturating irradiance (Ek)] and supported the temperature range findings in terms of the tolerance of the specimens in the low temperatures up to 31.0 °C. For all photochemical measurements assessed, the holobiont values tended to be somewhat higher than those for the symbionts, with the exception of Ek, possibly indicating a tight coupling in the host-symbiont response during photosynthesis. Chlorophyll a (<μ>g/foram) was negatively correlated with temperature (r = -0.37, p < 0.001) in Experiments 1 and 2. However, in all 3 experiments, chlorophyll a was variable, suggesting a high degree of individual variability in A. angulatus and the ability to acclimate. Some differences observed among treatments may be related to differences in seasons when the specimens were collected and in length of time in culture prior to experiments. Holobiont median rETR light curve trends and photophysiological derived parameters recorded median Ek ranges of ~100-150 <μ>mol photons m-2 s-1, observed ETRmax light intensities of ~200 <μ>mol photons m-2 s-1 and photoinhibition, induced by increasing irradiance intensities, which occurred > 500 <μ>mol photons m-2 s-1. These light curve trends and derived parameters generally supported previous photosynthesis O2 and CO2 gas production studies of A. angulatus. The differences in responses associated with acclimation should be considered in design of future experimental studies. This was the first known physiological study of the viable temperature range and photobiology of A. angulatus using chlorophyll fluorometry methods. Though commonly found in Caribbean and Atlantic waters ranging from 14.0 - 31.0 °C, these results indicate a wider thermal-tolerance range for A. angulatus than was previously known. Keywords: Foraminifera, Chlamydomonas sp., PAM fluorometry, photosynthesis, algal symbiosis