The overall objective of this PhD thesis research is to understand and control phase separation in mixed perfluorinated fatty acid-phospholipid surfactant systems that have applications as pulmonary surfactant (PS) mixtures, with an ultimate view of controlling film composition, morphology and mechanical properties. In this context the interaction between perfluorooctadecanoic acid (C18F), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), the major component of native PS extract, and 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) has been explored in Langmuir monolayers and Langmuir–Blodgett (LB) films using a combination of atomic force microscopy (AFM), fluorescence microscopy (FM) and Brewster angle microscopy (BAM) measurements. Thermodynamic and morphological studies of binary and ternary mixed films made of C18F, DPPC and DPPG indicated that both the phospholipids and C18F were miscible over a wide range of compositions. The mixed phospholipid-C18F films contained multimolecular aggregates that were highly enriched in the phospholipids. Furthermore, it was found that the magnitude of the DPPC-C18F interaction could be modulated by altering the concentration of sodium ions in the underlying subphase. Using a highly simplified lung mimic fluid (pH 7.4, 150mM NaCl), DPPC and C18F became fully immiscible. Moreover, the performance characteristics of the mixed films demonstrated the usefulness of C18F as an additive for PS formulations. The effectiveness of a PS protein mimicking peptide was evaluated against DPPC to allow comparison with previous measurements of DPPC-C18F mixed system. The mixing thermodynamics of the peptide and DPPC in Langmuir monolayer implied a repulsive interaction between the film components. The hysteresis response of the mixed monolayer films indicated that the lipid-protein mixture improved the re-spreading of DPPC films. Moreover, molecular-level organization of the mixed films explored by both FM and BAM confirmed the formation of liquid-expanded DPPC domains in the presence of minute amount of the peptide. In order to obtain a thorough understanding of the effect of the deposition process and surfactant tail polarities on the interfacial behavior of perfluorocarbon-hydrocarbon mixed monolayer films, both BAM and AFM measurements of arachidic acid (C20) with perfluorotetradecanoic acid (C14F) and palmitic acid (C16) with C18F mixed monolayer were performed. These measurements revealed that film morphology was minimally perturbed upon its deposition onto solid substrates. Coarse grained molecular dynamics (MD) simulations of films comprised of DPPC molecules with tails of various polarities suggested that the phase separation between the monolayer components could be controlled by varying surfactant tail polarities.

To effectively design systems of microchannels it is necessary for scientists and engineers to understand thermal transport characteristics of microchannels. To experimentally determine the convective heat transfer coefficient of microchannels it is necessary to measure both the bulk and surface temperature fields. This investigation aims to develop a technique, named Total Internal Reflection Fluorescent Micro-Thermometry (TIR-FMT), to measure the temperature of water within several hundred nanometers of a wall--effectively, the surface temperature of the wall. In TIR-FMT, an evanescent-wave is generated in the water near the wall. The intensity of this evanescent-wave decays exponentially with distance from the wall. A fluorophore if illuminated by the evanescent-wave can absorb a photon. Excited fluorophores subsequently emit red-shifted photons, which are called fluorescence. The probability of a fluorescent emission is temperature-dependent. Therefore, by monitoring the intensity of the fluorescence a correlation can be made to the temperature of the region of illumination. Using the TIR-FMT technique the temperature dependence of the fluorescence intensity from buffered fluorescein (pH=9.2) was determined to be 1.35%/C. TIR-FMT can be used to measure the temperature of a fluorophore solution within 600 nm of a wall across a temperature range of 12.5-55C. The average uncertainties (95% confidence) of the temperature measured was determined to be 2.3C and 1.5C for a single 1.5x1.5 and #956;m pixel and the entire 715x950 and #956;m viewfield. By spatial averaging, average uncertainties of 2.0C and 1.8C were attained with spatial resolutions of 16x16 and 100x100 and #956;m, respectively.

In numerous Gram-negative and Gram-positive bacteria as well as in Archaea SL proteins form the outermost layer of the cell envelope. SL (glyco)monomers self-assemble with oblique (p2), tetragonal (p4), or hexagonal (p3, p6) symmetries [12]. SL subunits interact with each other and with the underlying cell surface by relatively weak non-covalent forces such as hydrogen-bonds, ionic bonds, salt-bridges or hydrophobic interactions. This makes them easy to isolate by applying chaotropic agents like urea and guanidine hydrochloride (GuHCl), chelating chemicals, or by changing the pH of the environment [10]. Upon dialysis in an ambient buffer monomers recrystallize into regular arrays that possess the forms of flat sheets, open ended cylinders, or spheres on solid substrates, at air-water intefaces and on lipid films, making them appealing for nanobiotechnological applications [3, 18]. The aim of this study was to investigate the structure, thermal stability, in vivo self-assembly process, recrystallization and metallization of three different recombinant SL proteins (SslA-eGFP, mSbsC-eGFP and S13240-eGFP) expressed in yeast S. cerevisiae BY4741 which could be further used in nanobiotechnological applications. In order to fulfill this aim, I investigated the in vivo expression of SL proteins (SslA, SbsC, S13240) tagged with eGFP (SL-eGFP) in the yeast S. cerevisiae BY4141. First, I characterized the heterologous expression of SL fusion constructs with growth and fluorescence measurements combined with Western blot analyses. Fluorescence microscopy investigations of overnight grown cultures showed that SslA-eGFP fusion protein was expressed as fluorescent patches, mSbsC-eGFP as tubular networks, and S13240-eGFP as hollow-like fibrillar network structures, while eGFP did not show any distinct structure Thermal stability of in vivo expressed SL-eGFP fusion proteins were investigated by fluorescence microscopy and immunodetection. In vivo self-assembly kinetics during mitosis and meiosis was the second main issue. In parallel, association of in vivo mSbsC-eGFP structures with the cellular components was of interest. A network of tubular structures in the cytosol of the transformed yeast cells that did not colocalize with microtubules or the actin cytoskeleton was observed. Time-resolved analysis of the formation of these structures during vegetative growth and sporulation was investigated by live fluorescence microscopy. While in meiosis ascospores seemed to receive assembled structures from the diploid cells, during mitosis surface layer structures were formed de novo in the buds. Surface layer assembly always started with the appearance of a dot-like structure in the cytoplasm, suggesting a single nucleation point. In order to get these in vivo SL assemblies stably outside the cells (in situ), cell distruption experiments were conducted. The tubular structures formed by the protein in vivo were retained upon bursting the cells by osmotic shock; however their average length was decreased. During dialysis, monomers obtained by treatment with chaotropic agents recrystallized again to form tube-like structures. This process was strictly dependent on calcium ions, with an optimal concentration of 10 mM. Further increase of the Ca2+ concentration resulted in multiple non-productive nucleation points. It was further shown that the lengths of the S-layer assemblies increased with time and could be controlled by pH. After 48 hours the average length at pH 9.0 was 4.13 µm compared to 2.69 µm at pH 5.5. Successful chemical deposition of platinum indicates the potential of recrystallized mSbsC-eGFP structures for nanobiotechnological applications. For example, such metalized protein nanotubes could be used in conductive nanocircuit technologies as nanowires.

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In the first part of this thesis, we study the kinetics of the complexation of a double-stranded DNA byNCp7 protein. To do this, we study the evolution of mechanical properties of DNA and its complexation by stretching the DNA/NCp7 complex with a optical trap. We observed that the persistence length of the complex decreases progressively during the complexation. Using astatistical model we describe the evolution of the flexibility of DNA complexed with NCp7. Our main result is that the fraction phi of base pairs that have reacted is not a linear function of time at low phi.We interpret our results assuming that the adsorption of NCp7 on DNA is highly cooperative. In the second chapter, we describe the dynamics of probe particles in a colloidal glassy suspension of Laponite. Laponite is a colloidal discoidal particle of 25 nm in diameter and 0.92 nm thick. We take advantage of evanescent wave microscopy, and follow the movement of fluorescent latex particles.Then we image these particles. We show that for a movement that has a single characteristic time scale, it is simply a linear function of time. We find that, what ever their size, the motion of probe particles can be described by a succession of two dynamic modes, where the fastest mode corresponds to the diffusion of particles in a viscoelastic fluid.

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HER2 breast cancer is an aggressive disease that occurs in 20 - 30% of the breast cancer population. Treatment for HER2 breast cancer includes use of an anti-HER2 monoclonal antibody, trastuzumab. Testing for HER2 is of critical importance due to the adverse side effects and substantial costs associated with this anti-HER2 treatment. Currently, two kinds of tests, Fluorescence In Situ Hybridization (FISH) and Immunohistochemistry (IHC), are FDA approved for determination of HER2 status in breast cancers. Clinical and non clinical factors that affect the choice HER2 test and the use of anti-HER2 therapy in breast cancer were analyzed using a data set containing information from six outpatient oncology clinics in the United States. The analysis showed that geographic location, cancer stage, and diagnosis date (pre- or post-publication of testing guidelines) have significant effects on choice of test. With regard to trastuzumab prescription, geographic location and HER2 status have significant effects on the prescription of trastuzumab. In addition, there was a non-significant trend for certain Medicare patients not to receive trastuzumab therapy. These findings indicate that disparities are present in breast cancer care based on geography and cancer stage, and highlight the importance of testing guidelines. The cost effectiveness of FISH vs. IHC was determined, by considering the financial and health-related costs associated with testing and subsequent treatment as well as the accuracy of each test. The results show that FISH is the optimal choice for HER2 testing and is more cost-effective than IHC.