Thesis (M. S.)--Biomedical Engineering, Georgia Institute of Technology, 2006. / Committee Chair: Zhu, Cheng; Committee Member: Babensee, Julia; Committee Member: Dustin, Michael; Committee Member: Garcia, Andres; Committee Member: Jo, Hanjoong; Committee Member: van der Merwe, Anton. Part of the SMARTech Electronic Thesis and Dissertation Collection. Non-Latin script record

Thesis (M. Sc.)--York University, 2001. Graduate Programme in Biology. / Typescript. Includes bibliographical references (leaves 106-118). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://wwwlib.umi.com/cr/yorku/fullcit?pMQ66399.

The embryonic stem cell (ESC) microenvironment contains various localized physical and biochemical cues to direct cell fate. Current approaches for microenvironmental regulation rely on restricting cell behaviour to control endogenous signals such as secreted ligands. This report presents a microfluidic device that can directly manipulate the removal of autoregulatory ligands from culture and control the activation of Signal Transducer and Activator of Transcription-3 (Stat3) in ESCs. Specifically, the response of Stat3 was measured under diffusive and convective mass transfer regimes. A Brownian dynamics algorithm was also developed to simulate ligand transport and predict cellular response under these conditions. Stat3 activation under perfusion culture was found to depend on flow rate and axial distance in the flow direction. Long-term perfusion also allowed for the formation of a sustained gradient of Stat3 activation that led to selective loss of ESC pluripotency. These results demonstrate the utility of microfluidic culture for stem cell bioengineering applications.

The embryonic stem cell (ESC) microenvironment contains various localized physical and biochemical cues to direct cell fate. Current approaches for microenvironmental regulation rely on restricting cell behaviour to control endogenous signals such as secreted ligands. This report presents a microfluidic device that can directly manipulate the removal of autoregulatory ligands from culture and control the activation of Signal Transducer and Activator of Transcription-3 (Stat3) in ESCs. Specifically, the response of Stat3 was measured under diffusive and convective mass transfer regimes. A Brownian dynamics algorithm was also developed to simulate ligand transport and predict cellular response under these conditions. Stat3 activation under perfusion culture was found to depend on flow rate and axial distance in the flow direction. Long-term perfusion also allowed for the formation of a sustained gradient of Stat3 activation that led to selective loss of ESC pluripotency. These results demonstrate the utility of microfluidic culture for stem cell bioengineering applications.

This work describes the results of experimental study of the flow of soft objects (microgels) through microchannels. This work was carried with the intention of building a fundamental biophysical model for the flow of neutrophil cells in microcirculatory system. In Chapter 1 we give a summary of the literature describing the flow of cells and “model cells” in microchannels. Paramount to this we developed methods to modify microchannels fabricated in poly(dimethyl siloxane) (PDMS). Originally, these microchannels could not be used to mimic biological microenvironments because they are hydrophobic and have rectangular cross-sections. We designed a method to create durable protein coatings in PDMS microchannels, as outlined in Chapter 3. Surface modification of the channels was accomplished by a two-step approach which included (i) the site-specific photografting of a layer of poly(acrylamide) (PAAm) to the PDMS surface and (ii) the bioconjugation of PAAm with the desired protein. This method is compatible with different channel geometries and it exhibits excellent longevity under shear stresses up to 1 dyn/cm. The modification was proven to be successful for various proteins of various molecular weights and does not affect protein activity. The microchannels were further modified by modifying the cross-sections in order to replicate cardiovascular flow conditions. In our work, we transformed the rectangular cross-sections into circular corss-sections. Microchannels were modified by polymerizing a liquid silicone oligomer around a gas stream coaxially introduced into the channel, as outlined in Chapter 3. We demonstrated the ability to control the diameter of circular cross-sections of microchannels. The flow behaviour of microgels in microchannels was studied in a series of experiments aimed at studying microgel flow (i) under electrostatic interactions (Chapter 4), (ii) binding of proteins attached to the microgel and the microchannel (Chapter 5) and (iii) under the conditions of varying channel geometry (Chapter 6). This work overall present’s new methods to study the flow of soft objects such as cells, in the confined geometries of microchannels. Using these methods, variables can be independently probed and analyzed.

Thesis (Ph.D.)--University of Melbourne, Howard Florey Institute and Dept. of Biochemistry and Molecular Biology, 2006. / Typescript. Includes bibliographical references (leaves 133-150).

Thesis (Ph. D.)--Northeastern University, 2008. / Title from title page (viewed Aug. 5, 2009). Graduate School of Arts and Sciences, Dept. of Chemistry and Chemical Biology. Includes bibliographical references.

Thesis (Ph. D.)--Rutgers University, 2007. / "Graduate Program in Neuroscience." Includes bibliographical references (p. 176-197).

Thesis (Ph. D.)--Rutgers University, 2008. / "Graduate Program in Neuroscience." Includes bibliographical references (p. 151-166).

A modelagem das interações entre macromoléculas e ligantes ainda se depara com diversos desafios na área de desenho de fármacos assistidos por computador. Apesar do crescimento da área, temas como a flexibilidade do receptor, funções de pontuação e solvatação ainda têm sido alvo de intensa investigação na comunidade científica. Com o objetivo de analisar a interação em milhares ou milhões de complexos, é imprescindível uma boa harmonização entre o custo computacional e a acurácia dos métodos computacionais que permitem a classificação de ligantes de acordo com a energia de interação. O LiBELa (Ligand Binding Energy Landscape) é um programa de docagem molecular com abordagem híbrida, ou seja, utiliza informações do ligante e do receptor durante o processo de docagem. Inicialmente, as características estéricas e eletrostáticas de um ligante de referência (cristalográfico, por exemplo) são utilizadas nos cálculos de similaridade e sobreposição, obtendo assim uma conformação inicial pré-otimizada do ligante testado. Em seguida, a energia de interação é minimizada no sítio ativo de receptor a partir de potenciais energéticos. Quatro funções de pontuação baseadas em campo de força foram testadas e otimizadas, compostas por potenciais de van der Waals, de Coulomb, e uma função empírica de solvatação denominada função de Stouten-Verkhivker (SV). A flexibilidade do sistema foi tratada através da geração de confôrmeros que amostram os graus de liberdade dos ligantes descritos como semi-rígidos e através de potenciais atenuados que suavizam a superfície de energia de interação, permitindo interações em distâncias interatômicas antes repulsivas. Como ponto de partida, os métodos implementados no programa LiBELa demonstraram resultados satisfatórios nos testes de cross- e self-docking, mostrando ser uma ferramenta eficiente em encontrar os modos de ligação cristalográficos de forma equivalente ou até melhor às dos programas comparados. Através de testes de enriquecimento nos conjuntos de dados DUD, DUDE e CM-DUD, foram otimizadas de forma sistemática as constantes dielétrica, do termo de solvatação, e dos termos de atenuação. Também foi realizado um paralelo entre as funções de pontuação, incluindo a atenuação e o termo de solvatação. Estes mesmos testes mostraram resultados superiores do LiBELa de 39% e 15% em comparação com um programa baseado puramente no receptor (DOCK 6.6), relativo à média da área sob a curva em escala semi-logarítmica nas bases de dados DUDE e DUD respectivamente. Apesar da função de solvatação SV implementada no LiBELa apresentar boa correlação com dados experimentais (r=0,72) e com o modelo Zou GB de solvatação (r=0,88), não apresentou correlação significativa com os métodos GB e PB implementados no pacote de programas disponível no AmberTools. Comparadas às funções de pontuação do LiBELa, as funções com correção para solvatação apresentaram pior enriquecimento, salvo alguns alvos específicos. Por fim, foram realizados ensaios de docagem molecular utilizando como alvo uma enzima β-galactosidase da família GH42, cuja estrutura fora resolvida em nosso grupo. Os resultados permitiram conclusões acerca de como o modo de ligação interfere na preferência de ligação entre dissacarídeos de ligações glicosídicas distintas, consistentes com dados experimentais de ensaios cinéticos de ligação. / Modeling the interactions between macromolecules and ligands still faces several challenges in the computer-aided drug design area. Despite the growth in the area, subjects such as receptor flexibility, scoring functions and solvation still have been widely explored in the scientific community. In order to analyze the interaction for thousands or millions of complexes, a good harmonization between the computational cost and the accuracy of the calculation methods in molecular docking programs is essential. LiBELa (Ligand Binding Energy Landscape) is a hybrid approach program that uses both ligand and receptor information for ligand docking. Initially, the steric and electrostatic characteristics from a reference binder (crystallographic, for example) are used to similarity and overlay calculations, thus obtaining an initial conformation of the ligand tested. Then, within the receptor´s active site, the interaction energy is minimized using energetic potentials. Four force field-based scoring functions were tested and optimized, composed of van der Waals and Coulomb potentials and an empirical solvation function called Stouten-Verkhivker (SV). Concerning the system flexibility, besides the confomers generation that sample the degrees of freedom for semi-rigid ligands, attenuated potentials smooth the energy surface allowing interactions between previously repulsive interatomic distances. As a starting point, LiBELa performed satisfactorily in the cross- and self-docking tests, showing that is an eficient tool to reproduce crystallographic binding modes equivalently to or even better than reference programs. Through enrichment of DUD, DUDE and CM-DUD datasets, the dielectric constant, solvation and softening terms were systematically optimized. It also allowed a parallel between scoring functions, including attenuation and solvation term. Finally, it revealed the LiBELa showed an enhancement of 39% and 15% as compared to the purely receptor-based program DOCK 6.6, relative to the mean of the area under the curve on a semi-logarithmic scale in the DUDE and DUD databases respectively. Although the SV solvation function implemented in LiBELa showed good correlations with experimental data (r = 0.72) and with the Zou GB / SA solvation method implemented in DOCK6 (r = 0.88), it did not show significant correlation with the GB/SA and PB/SA methods implemented in AmberTools. Comparing all the LiBELa tested scoring functions, those including solvation correction showed worse enrichments, except for some specific targets. Finally, molecular docking experiments using LiBELa were conducted with a β-galactosidase from GH42 family, whose structure was solved in our group. The results allowed conclusions concerning how the binding mode interferes the preference for some disaccharides of distinct glycosidic bonds, consistent with experimental data from kinetic assays.