<p>The present thesis concerns theoretical studies of molecular interactions investigated by infrared and X-ray spectroscopic techniques, with emphasis on using these two techniques combined in pump-probe experiments. Four main types of studies are addressed: the use of near-edge X-ray absorption fine structure spectra (NEXAFS) to manifest through-bond and through-space interactions; the role of hydrogen bonding in the formation of X-ray photoelectron spectra as evidenced by simulations of the water dimer; the development of theory, with sample applications, for infrared X-ray pump-probe spectroscopy; and molecular dynamics simulations of light-induced fragmentation of water clusters.</p><p>Ab initio calculations indicate that NEXAFS spectra give direct information about the through-bond and through-space interactions between vacant non-conjugated π* orbitals. It is found out that the X-ray photoelectron spectrum of the water dimer differs dramatically from the monomer spectrum in that two bands are observed, separated by the chemically shifted ionization potentials of the donor and the acceptor. The hydrogen bond is responsible for the anomalously strong broadening of these two bands. The studies show that X-ray core electron ionization of the water dimer driven by an infrared field is a proper technique to prove the proton transfered state contrary to conventional X-ray photoelectron spectroscopy. </p><p>The physical aspects of the proposed new X-ray spectroscopic method - phase sensitive Infrared - X-Ray Pump-Probe Spectroscopy - are examined in detail using the wave packet technique in three applications; the NO molecule and the dynamics of proton transfer in core ionized water dimer and glyoxalmonoxime. It is found out that the phase of the infrared pump field strongly influences the trajectory of the nuclear wave packet on the ground state potential, which results in a phase dependence of the X-ray pump-probe spectra. A proper choice of the delay time of the X-ray pulse allows the direct observation of the X-ray transition in the proton transfered well of the core excited potential. It is found out that the glyoxalmonoxime molecule possesses an important feature; proton transfer accompanied by core hole hopping. Special attention is paid to the quantum control of the populations of vibrational level which is of crucial importance to shape the wave packet of desirable size.</p><p>The wave packet technique becomes computationally very expensive when the number of nuclear degrees of freedom is large. Molecular dynamics is used instead in studies of light-induced nuclear kinetics in the water hexamer cluster. We predict a novel mechanism of the mechanical action of light on atoms and molecules. This mechanism is based on the rectification of the Lorentz force, which gives a unique opportunity of direct site selective mechanical action of light on atoms and molecules inside large systems like clusters or biomolecules.</p>

<p>The work presented in the thesis concerned theoretical study of light-matter nonlinear interactions. Two important aspects of such interactions have been examined, namely the nonlinear optical properties of a series of organic charge transfer molecules in solutions induced by the laser light and the propagation of the ultrafast high power laser through the nonlinear molecular medium.</p><p>Special attention has been paid to understand the solvent effects on the two-photon absorption of a symmetrical diamino substituted distyrylbenzene chromophore, for which time-dependent density functional theory in combination with polarizable continuum model (PCM) have been employed. The dielectric medium alone has a rather small effect both on the bond length alternation and on the one-photon absorption spectrum, but noticeable effects on the two-photon absorption cross section. Both one- and two-photon absorptions are found to be extremely sensitive to the planarity of the molecule. Our calculations indicate that the experimentally observed anomalous solvent effect on the two-photon absorption of dialkylamino substituted distyrylbenzene chromophores can not be attributed to the intrinsic properties of a single molecule and its interaction with solvents. With the same theoretical approaches, two-photon absorption properties of interacting polar chromophores have been investigated to examine the validity of the widely used exciton model. Our first principles calculations have shown that the exciton model offers a conceptually simple interpretation for experimental observations, but is lack of predictability.</p><p>The second part of the thesis is to investigate the propagation of ultrashort laser pulse through a one-dimensional asymmetric organic molecular medium by solving full Maxwell-Bloch equations using predictor-corrector finite-difference time-domain method. It focuses on the supercontinuum generation of spectra and the formation of attosecond pulses. It is shown that the supercontinuum generation is strongly modulated by both area and width of the pulse, which results from the interference between the splitting pulses in time-domain and is the implication of the time-energy uncertainty relation. The presence of permanent dipole moment in molecular medium has noticeable effects on the supercontinuum generation. Our calculations show that a well-shaped 132 attosecond pulse can be generated from a two femtosecond incoming pulse under certain conditions. Influences of carrier-envelope phase and time-dependent ionization on the spectral and temporal evolutions of the ultrashort pulses have also been discussed.</p>

<p><i>In this thesis, quantum chemical methods are used to investigate enzymatic reaction mechanisms. The Density functional theory, in particular the hybrid B3LYP functional, is used to model two enzymes belonging to the tautomerase superfamily; 4-Oxalocrotonate Tautomerase (4-OT) and cis-Chloroacrylic Acid Dehalogenase (cis-CAAD). The methodology is presented and new mechanistic insights for the two enzymes are discussed.</i></p><p><i>For 4-OT, two different models are built and the potential energy curves are computed. This allows the methodology to be evaluated. The results give new insight into the energetics of the 4-OT reaction, indicating that the charge-separated intermediate is quite close in energy to the reactant species. The models also make it possible to perform in silico mutations to investigate the role of active site groups. Excellent agreement is found between the calculations and site-directed mutagenesis experiments, further substantiating the validity of the models.</i></p><p><i>For cis-CAAD, the uncatalyzed reaction is first considered and excellent agreement is found between the calculated barrier and the measured rate constant. The enzymatic reaction is then studied with a quite large active site model and a reaction mechanism is proposed.</i></p>

<p>The recombinant product is secreted to the periplasm in many processes where<em> E. coli</em> is used as host. One drawback with secretion is the undesired leakage of the periplasmic products to the medium.</p><p>The aim of this work was to find strategies to influence the periplasmic retention of recombinant products. We have focused on the role of the specific growth rate, a parameter that is usually controlled in industrial bioprocesses. The hypothesis was that the stability of the outer membrane in <em>E. coli </em>is gained from a certain combination of specific phospholipids and fatty acids on one side and the amount and specificity of the outer membrane proteins on the other side, and that the specific growth rate influences this structure and therefore can be used to control the periplasmic retention.</p><p>We found that is possible to control the periplasmic retention by the growth rate. The leakage of the product increased as the growth rate increased. It was however also found that a higher growth rate resulted in increased productivity. This resulted in equal amounts of product inside the cells regardless of growth rate.</p><p>We also showed that the growth rate influenced the outer membrane composition with respect to OmpF and LamB while OmpA was largely unaffected. The total amount of outer membrane proteins decreased as the growth rate increased. There were further reductions in outer membrane protein accumulation when the recombinant product was secreted to the periplasm. The lowered amount of outer membrane proteins may have contributed to the reduced ability for the cell to retain the product in the periplasm.</p><p>The traditional way to control the growth rate is through a feed of substrate in a fed-batch process. In this work we used strains with a set of mutations in the phosphotransferase system (PTS) with a reduced uptake rate of glucose to investigate if these strains could be used for growth rate control in batch cultivations without the use of fed-batch control equipment. The hypothesis was that the lowering of the growth rate on cell level would result in the establishment of fed-batch similar conditions.</p><p>This study showed that it is possible to control the growth rate in batch cultivations by using mutant strains with a decreased level of substrate uptake rate. The mutants also produced equivalent amounts of acetic acid as the wild type did in fed-batch cultivation with the same growth rate. The oxygen consumption rates were also comparable. A higher cell density was reached with one of the mutants than with the wild type in batch cultivations. It is possible to control the growth rate by the use of the mutants in small-scale batch cultivations without fed-batch control equipment.</p>

Density functional theory (DFT) has become the leading method in calculating theelectronic structures and properties from first principles. In practical applicationsof DFT in the frame work of Kohn-Sham (KS) method, an approximate exchange-correlation functional has to be chosen. Hence, the success of a DFT calculationcritically depends on the quality of the exchange-correlation functional.This thesis focuses on the development and validation of the so-called dou-bly hybrid density functionals (DHDFs). DHDFs present a new generation offunctionals, which not only have a non-local orbital-dependent component in theexchange part, but also incorporate the information of unoccupied orbitals in thecorrelation part. I will first give an overview of modern DFT in the introductorychapters, emphasizing the theoretical bases of a newly developed DHDF, XYG3.I will then present further examination of XYG3 and new development on top ofXYG3, leading to XYG3o and XYG3s. Attempts have also been made to extractband structure information of a periodic system from cluster model calculations. / QC 20110607

Microfluidic chips have become a powerful tool in research where biological cells are processed and/or analyzed. One method for contactless cell manipulation in microfluidic chips that has gained an increasing amount of attention the last decade is ultrasonic standing wave (USW) technology. This Thesis explores the biocompatibility of USW technology applied to microfluidic chips, and presents a novel USW-based method for serial processing and accurate characterization of living cells. The biocompatibility has been investigated by measuring the proliferation rate of cells after they had been trapped and aggregated inside a chip by ultrasound. No negative influence was observed after continuous exposure to 0.85 MPa pressure amplitudes for up to 75 min. Furthermore, the heat generation in the fluid channel caused by the ultrasound has been measured and used in a regulation scheme where the temperature can be controlled around any relevant temperature (e.g. 37‰) with ±0.1‰ accuracy for more than 12 hours. The proliferation rate and temperature investigations suggest that USW technology applied to microfluidic chips is a biocompatiblemethod useful for long-term handling of living cells. We have introduced a new concept of contactless ultrasonic ”caging” of single cells or small aggregates of cells. These cages are channel segments in the microfluidic chips that are geometrically designed to resonate at one or several actuation frequencies. The actuation is performed remotely by up to five external frequency specific wedge transducers, where each transducer produces a localized and spatially confined standing wave with a specific orientation of its corresponding radiation force field. By multi-frequency actuation, sophisticated and flexible force fields are realized by both overlapping and separated single fields. The Thesis describes two different cages: A sub-mm ”micro-cage” for tree-dimensional manipulationof single cells, and a 5-mm ”mini-cage” for selective retention of small cell aggregates (up to approx. 10^3 cells) from a continuously feeding sample flow. Finally,our microfluidic chips were also designed to be compatible with high-resolution optical microscopy. We have demonstrated sub-μm-resolution confocal fluorescence and trans-illumination microscopy imaging of ultrasonically caged living cells. / QC 20100811

Poly (ethylene glycol) (PEG) hydrogels have been used extensively in biological and tissue engineering, because of their outstanding biocompatibility and processability. However, it is not yet possible to process soft materials like PEG hydrogels with the requisite precision and throughput needed to recapitulate macroscopic biological tissue with control over every hierarchical scale. In this study, porous PEG hydrogels are processed by a phase separation method and patterned in a single photolithographic step. The thermodynamics of the temperature triggered spinodal decomposition of a ternary mixture of water, salt, and polymer are studied resulting in a ternary phase diagram and a spinodal temperature plot. Importantly, the state of porosity can be frozen by exposing the hydrogel to UV light to form a crosslinked hydrogel network. The average pore size can be tuned by changing delay between the application of heat and UV exposure. By utilizing grey-scale photomasks, a single process can be used to define regions of pure hydrogel, porous hydrogel with a programmed average pore size, and blank substrate with no hydrogel. In addition to representing a combination of a top-down and a bottom-up processes that enables the realization of complex samples, the simplicity of this process and the versatility of the resultant patterns could provide a useful capability for the definition of hydrogel samples for the development of advanced biomaterials.

The molecular composition and hierarchical structure of bone tissue are important determinants of bone strength and fragility. Vibrational spectroscopic techniques have frequently been used to investigate bone composition, including attenuated total reflection (ATR) and Fourier transform infrared (FTIR) imaging spectroscopy. However, with these approaches, the spatial resolution of analysis is limited to several micrometers, which cannot capture properties at the nanoscale level of mineralized collagen fibrils (~500 nm), the building blocks of bone. Recent advances in optical photothermal infrared (O-PTIR) spectroscopy allows investigation of bone composition with submicron spatial resolution. In this thesis, we hypothesize that higher resolution information related to bone composition will provide new insights into factors underlying bone function. In two Aims, we: 1. Investigated whether O-PTIR-derived spectral parameters correlated to standard ATR spectral data from homogenized serially demineralized bone samples. 2. Examined whether O-PTIR-derived spectral parameters, including nanoscale heterogeneity of tissue, contribute to prediction of bone stiffness. Bovine tibial cortical bone was homogenized into powder and serially demineralized using EDTA. ATR and O-PTIR spectra were collected at six timepoints (n=6) over 5 days. Femoral neck bones from human donor cadaver tissues were sliced into 1mm sections. Line scans of 10 different trabecula were collected for each sample, with a total of 15 samples. Spectral images were collected from the intact femoral neck samples, which also had sex, age, and BMI information, as well as experimentally-determined stiffness parameters, associated with them. There was a correlation of R = 0.96 between the 2nd derivative phosphate (PO4) /amide II peak intensity ratio for ATR to O-PTIR. Principal component analysis (PCA) yielded insight into the variance between O-PTIR and ATR spectra being mainly attributable to the phosphate absorbance peak (PO4). We also found that O-PTIR nanoscale assessment of bone parameters was more sensitive to the acid phosphate peak (HPO4) associated with newly formed bone. Partial least squares (PLS) regression analysis showed that combining multiple O-PTIR parameters (HPO4 content and heterogeneity, collagen integrity and CO3 content) can significantly predict whole-bone proximal femoral stiffness with an R2 of 0.74. Overall, this study highlights a new application of O-PTIR spectroscopy to assess bone composition at a submicron scale, which may provide new insights into molecular-level factors underlying bone mechanical competence. / Chemistry

Wound healing is a complex, dynamic process that needs to be orchestrated in an orderly manner, involving different cell types, cytokines, and growth factors as well as extracellular matrix (ECM) interactions to avoid complications (e.g. scarring, chronic wounds, and impaired functionality). Current strategies for management of full thickness wounds are limited by material expense, limited availability of allograft tissue, autograft donor site morbidity, and even ethical problems associated with animal derived matrix components. To avoid cost, ethical, and even safety issues, there is renewed interest in using natural ingredients to construct advanced scaffolds for wound healing. At the cutting edge of the new field of regenerative medicine, demonstrated here is a scaffold based on soy protein. The second generation of soy based scaffold is constructed at the nanometer level to provide an ideal environment for cell interaction, growth, and development through a modified process termed ‘electro-processing’. The electro-processing developed here uses only water, low pressure air, and a very low current high voltage source improving not just the safety of the product, but also improving the manufacturing process. The proteins have also been indicated to release pro-healing cue to the surrounding tissue. Processing the natural soy protein to improve solubility has even enabled the new scaffolds to be generated without any harmful solvent and at rates many times faster than those previously demonstrated and at increased quality. The novel soy based electro-processed bioactive wound fabrics have been demonstrated successfully in vitro and in preliminary in vivo testing. These scaffolds have shown to be biocompatible, degradable and to improve healing quality compared to Tegaderm dressing in a rat full thickness excision model. / Bioengineering

While mechanical, homograft and bio-prosthetic valves have been used in patients for many decades and have made significant improvements in patient morbidity, there is still a distinct need to overcome their limitations. Recently, emerging elastomer heart valves have been shown to be able to better re-create the flow physics of native heart valves, resulting in preferable hemodynamic responses. Unfortunately, elastomers such as silicone are prone to structural failure, which drastically limits their applicability towards the development of valve prosthesis. In order to produce a mechanically more robust silicone substrate, we reinforced it with graphene nanoplatelets (GNPs). Cytotoxicity and hemocompatibility tests revealed that the incorporation of GNPs did not adversely affect cell proliferation or augment adhesion of platelets on the surface of the composite materials. The ECM valves showed good hydrodynamic properties and favorable acute performance compared to a commercially available valve. We conclude that both the Graphene reinforce silicone and the ECM is useful and warrants further evaluation as aortic valve substitutes.