Micro/nano-patterning of supported lipid bilayers (SLBs) has shown considerable potential for addressing fundamental biophysical questions about cell membrane behavior and the creation of a new generation of biosensors. Herein are presented several novel lithographic methods for the size-controlled patterning of SLBs from the microscale to the nanoscale. Using these methods, chemically distinct types of phospholipid bilayers and/or Escherichia Coli (E. Coli) membranes can be spatially addressed on a single microchip. These arrays can, in turn, be employed in the studies of multivalent ligand-receptor interactions, enzyme kinetics, SLBs size limitation, and membrane-associated species separation. The investigations performed in the Laboratory for Biological Surface Science include the following projects. Chapters II and III describe the creation of lab-on-a-chip based platforms by patterning SLBs in microfluidic devices, which were employed in high throughput binding assays for multivalent ligand-receptor interactions between cholera toxin B subunits (CTB) and ganglioside GM1. The studies on the effect of ligand density for multivalent CTB-GM1 interactions revealed that the CTB-GM1 binding weakened with increasing GM1 density. Such a result can be explained by the clustering of GM1 on the supported phospholipid membranes, which in turn inhibits the binding of CTB. Chapter IV characterizes the enzymatic activity of phosphatase tethered to SLBs in a microfluidic device. Higher turnover rate and catalytic efficiency were observed at low enzyme surface densities, ascribing to the low steric crowding hindrance and high enzyme fluidity, as well as the resulting improvement of substrate accessibility and affinity of enzyme catalytic sites. Chapter V presents sub-100 nm patterning of supported biomembranes by atomic force microscopy (AFM) based nanoshaving lithography. Stable SLBs formed by this method have a lower size limit of ~ 55 nm in width. This size limit stems from a balance between a favorable bilayer adhesion energy and an unfavorable bilayer edge energy. Finally, chapter VI demonstrates the electrophoretic separation of membrane-associated fluorophores in polymer-cushioned lipid bilayers. This electrophoretic method was applied to the separation of membrane proteins in E. Coli ghost membranes.

In vertebrates, the embryonic hindbrain is transiently subdivided along its anterior-posterior (A-P) axis into 8 well defined segments termed rhombomeres (r1-8). Each rhombomere represents a true cellular compartment in transcriptional profile, lineage restriction and neuronal organization. Thus, the vertebrate hindbrain provides a beautiful model for studying mechanisms of anterior-posterior patterning, signal transduction and interpretation, initiation and maintenance of transcriptional profiles, cell sorting and border formation. The Kreisler/MafB gene, which encodes a basic leucine zipper (bZIP) transcription factor that regulates some Hox genes, is one of the first genes to be expressed segmentally in the hindbrain, and is subject to a dynamic and complex regulatory process. However, unlike the Hox genes, Kreisler/MafB is not located within a large cluster of genes and therefore provides a simple system for dissecting the molecular mechanisms involved in hindbrain compartmentalization. In dissecting the mechanisms that govern Kreisler/MafB regulation, we have identified the S5 regulatory element that directs early MafB expression in the future r5-r6 domain. We have found a binding site within S5 that is specific for the Variant Hepatocyte Nuclear Factor 1 (vHNF1) to be essential, but not sufficient for early induction of r5-r6-specific expression. Thus, early inductive events that initiate MafB expression are clearly distinct from later acting ones that modulate its expression levels. Using mouse mutants, we have shown that MafB is dependent on the M33 polycomb protein and other mechanisms of chromatin remodeling. We then utilized transgenic flies and mice as well as binding assays to identify and validate a PcG/trxG response element (PRE), PRE1 which acts to reorganize the surrounding chromatin, regulating S5-dependent expression. To our knowledge, PRE1 is the first validated vertebrate PcG/trxG response element. Thus, PRE1 provides a springboard for further exploration of the mechanisms governing chromatin remodeling.

In vertebrates, the embryonic hindbrain is transiently subdivided along its anterior-posterior (A-P) axis into 8 well defined segments termed rhombomeres (r1-8). Each rhombomere represents a true cellular compartment in transcriptional profile, lineage restriction and neuronal organization. Thus, the vertebrate hindbrain provides a beautiful model for studying mechanisms of anterior-posterior patterning, signal transduction and interpretation, initiation and maintenance of transcriptional profiles, cell sorting and border formation. The Kreisler/MafB gene, which encodes a basic leucine zipper (bZIP) transcription factor that regulates some Hox genes, is one of the first genes to be expressed segmentally in the hindbrain, and is subject to a dynamic and complex regulatory process. However, unlike the Hox genes, Kreisler/MafB is not located within a large cluster of genes and therefore provides a simple system for dissecting the molecular mechanisms involved in hindbrain compartmentalization. In dissecting the mechanisms that govern Kreisler/MafB regulation, we have identified the S5 regulatory element that directs early MafB expression in the future r5-r6 domain. We have found a binding site within S5 that is specific for the Variant Hepatocyte Nuclear Factor 1 (vHNF1) to be essential, but not sufficient for early induction of r5-r6-specific expression. Thus, early inductive events that initiate MafB expression are clearly distinct from later acting ones that modulate its expression levels. Using mouse mutants, we have shown that MafB is dependent on the M33 polycomb protein and other mechanisms of chromatin remodeling. We then utilized transgenic flies and mice as well as binding assays to identify and validate a PcG/trxG response element (PRE), PRE1 which acts to reorganize the surrounding chromatin, regulating S5-dependent expression. To our knowledge, PRE1 is the first validated vertebrate PcG/trxG response element. Thus, PRE1 provides a springboard for further exploration of the mechanisms governing chromatin remodeling.

Gastrulation is one of the most critical events of embryogenesis, generating the three primary germ layers (definitive endoderm, mesoderm, and ectoderm) and establishing the embryonic body plan. The definitive endoderm, which generates the lungs, liver, pancreas, and digestive tract, has become a tissue of particular interest in recent years. Understanding definitive endoderm formation and patterning will greatly aid progress in the in vitro differentiation of embryonic stem cells to definitive endoderm for use in treatment of diseases such as diabetes and hepatitis as an alternative for whole organ replacement. Gene targeting studies have demonstrated a critical role for the Nodal signaling pathway and the forkhead transcription factors Foxh1 and Foxa2 in specification of a group of cells referred to as the anterior primitive streak (APS). However, the transcriptional targets of Foxh1 and/or Foxa2 other than Nodal that regulate specification of this group of cells are currently unknown. Fate mapping and lineage tracing experiments have shown the APS to be the source of the definitive endoderm. However, many questions regarding specification and patterning of the definitive endoderm remain. The study of this tissue has been hampered by the lack of genetic markers specific for the definitive endoderm as many of the current markers, including Cerl, Foxa2, and Sox17, are also expressed in the visceral endoderm, an extraembryonic tissue. To further investigate the role of Foxh1 in specification of the anterior primitive streak and to address the lack of genetic markers for the definitive endoderm we performed expression profiling on post-implantation mouse embryos using Affymetrix™ GeneChips®. From this analysis we identified and characterized a novel marker of the mouse definitive endoderm. Examination of this, and other, novel endoderm markers in Foxh1 and Foxa2 deficient mouse embryos revealed that contrary to current models of definitive endoderm formation, we find some definitive endoderm is formed in these mutants. Specifically, specification of the midgut and hindgut definitive endoderm is largely unaffected, while foregut formation is severely affected. These results suggest that the formation of the midgut and hindgut definitive endoderm populations is independent of the anterior primitive streak and separate from the foregut definitive endoderm. This represents a major insight into the mechanisms regulating endoderm formation and patterning.

En els darrers anys, el desenvolupament de nous mètodes per a la fabricació de xarxes ordenades de nanoestructures magnètiques (litografia magnètica) ha esdevingut un camp de recerca de gran interès. Això es deu tant per l'ampli ventall d'aplicacions tecnològiques que se'n deriven de les estructures magnètiques de grandària submicromètrica (biomedicina, gravació magnètica, ...), com per raons de caire més fonamental, ja que sovint el comportament magnètic d'aquests materials a escala nanomètrica és diferent del corresponent als materials massissos. Els aliatges de Fe60Al40 (percentatge atòmic) i els acers inoxidables austenítics posseeixen una combinació de propietats estructurals i magnètiques que els converteix en materials amb cert potencial per a ser litografiats magnèticament. Des d'un punt de vista magnètic, mentre que els aliatges de Fe60Al40 ordenats atòmicament són paramagnètics a temperatura ambient, els aliatges de Fe60Al40 desordenats a nivell atòmic presenten un comportament ferromagnètic. Pel que fa als acers inoxidables austenítics, a partir de deformació mecànica, es pot induir la transformació en estat sòlid de la fase austenita (paramagnètica) a la fase martensita (ferromagnètica) en aquests aliatges ferris. A més, els processos de nitruració a temperatura moderada en acers inoxidables austenítics permeten transformar parcialment la fase austenita en la fase "austenita expandida", que és una solució sòlida sobresaturada de nitrogen que presenta un comportament ferromagnètic. Aquesta Tesi està basada en la generació de xarxes ordenades d'entitats ferromagnètiques a escala micro/nanomètrica, dins d'una matriu paramagnètica, en la superfície d'aliatges de Fe60Al40 i acers inoxidables austenítics. Aquest propòsit s'assoleix aprofitant les transicions magnètiques que tenen lloc en aquests materials després de sotmetre'ls a processos de deformació plàstica local (nanoindentació) i irradiació controlada amb ions (αs de feixos d'ions focalitzats i irradiació amb ions de gasos nobles a través de màscares en el cas del Fe60Al40 i processos de nitruració a través de màscares d'irradiació en els acers inoxidables austenítics). Cal esmentar que també s'ha dut a terme un estudi detallat de les modificacions a nivell estructural, mecànic i magnètic que ocorren en aquests materials una vegada s'han deformat mecànicament o irradiat amb ions. / In recent years, intense research is being pursued in the development of novel methods for the fabrication of arrays of ordered magnetic nanostructures. This is motivated, in part, by the technological applications of sub-micron magnetic structures, ranging from biomedicine to recording media, but it is also due to fundamental scientific reasons, since the behavior of magnetic materials at this length scale is often significantly different from that in the bulk. Fe60Al40 (at. %) alloys and austenitic stainless steels show an interesting combination of magnetic and structural properties, which makes them turn into potential candidates to be magnetically patterned. Namely, from the magnetic point of view, whereas atomically ordered Fe60Al40 (at. %) is paramagnetic at room temperature, disordered Fe60Al40 becomes ferromagnetic. Concerning austenitic stainless steels, due to mechanical deformation, a phase transformation from the paramagnetic austenite phase to the ferromagnetic martensite phase can occur in these ferrous alloys. In addition, nitriding of austenitic stainless steels at moderate temperatures is able to partially transform the austenite phase into the supersaturated nitrogen solid solution, often called in the literature "expanded austenite" phase, which shows ferromagnetic behavior. This Thesis is mainly focused on the generation of ordered arrays of micro/nanoscaled ferromagnetic entities (i.e., magnetic patterning), embedded in a paramagnetic matrix, at the surface of either Fe60Al40 (at. %) alloys or austenitic stainless steels. This is accomplished by taking advantage of the magnetic transitions which occur in these alloys upon local plastic deformation (nanoindentation) and controlled ion irradiation (focused ion beam and broad beam noble gas ion irradiation through shadow masks in FeAl alloys and ion beam nitriding through shadow masks in austenitic stainless steels). Furthermore, a detailed study of the structural, mechanical and magnetic changes which take place in these materials upon either mechanical deformation or ion irradiation is presented.

Expanding the optical lithography to 32-nm node and beyond is impossible using existing single exposure systems. As such, double patterning lithography (DPL) is the most promising option to generate the required lithography resolution, where the target layout is printed with two separate imaging processes. Among different DPL techniques litho-etch-litho-etch (LELE) and self-aligned double patterning (SADP) methods are the most popular ones, which apply two complete exposure lithography steps and an exposure lithography followed by a chemical imaging process, respectively. To realize double patterning lithography, patterns located within a sub-resolution distance should be assigned to either of the imaging sub-processes, so-called layout decomposition. To achieve the optimal design yield, layout decomposition problem should be solved with respect to characteristics and limitations of the applied DPL method. For example, although patterns can be split between the two sub-masks in the LELE method to generate conflict free masks, this pattern split is not favorable due to its sensitivity to lithography imperfections such as the overlay error. On the other hand, pattern split is forbidden in SADP method because it results in non-resolvable gap failures in the final image. In addition to the functional yield, layout decomposition affects parametric yield of the designs printed by double patterning. To deal with both functional and parametric challenges of DPL in dense and large layouts, EDA solutions for DPL are addressed in this thesis. To this end, we proposed a statistical method to determine the interconnect width and space for the LELE method under the effect of random overlay error. In addition to yield maximization and achieving near-optimal trade-off between different parametric requirements, the proposed method provides valuable insight about the trend of parametric and functional yields in future technology nodes. Next, we focused on self-aligned double patterning and proposed layout design and decomposition methods to provide SADP-compatible layouts and litho-friendly decomposed layouts. Precisely, a grid-based ILP formulation of SADP decomposition was proposed to avoid decomposition conflicts and improve overall printability of layout patterns. To overcome the limited applicability of this ILP-based method to fully-decomposable layouts, a partitioning-based method is also proposed which is faster than the grid-based ILP decomposition method too. Moreover, an A∗-based SADP-aware detailed routing method was proposed which performs detailed routing and layout decomposition simultaneously to avoid litho-limited layout configurations. The proposed router preserves the uniformity of pattern density between the two sub-masks of the SADP process. We finally extended our decomposition method for double patterning to triple patterning and formulated SATP decomposition by integer linear programming. In addition to conventional minimum width and spacing constraints, the proposed decomposition method minimizes the mandrel-trim co-defined edges and maximizes the layout features printed by structural spacers to achieve the minimum pattern distortion. This thesis is one of the very early researches that investigates the concept of litho-friendliness in SADP-aware layout design and decomposition. Provided by experimental results, the proposed methods advance prior state-of-the-art algorithms in various aspects. Precisely, the suggested SADP decomposition methods improve total length of sensitive trim edges, total EPE and overall printability of attempted designs. Additionally, our SADP-detailed routing method provides SADP-decomposable layouts in which trim patterns are highly robust to lithography imperfections. The experimental results for SATP decomposition show that total length of overlay-sensitive layout patterns, total EPE and overall printability of the attempted designs are also improved considerably by the proposed decomposition method. Additionally, the methods in this PhD thesis reveal several insights for the upcoming technology nodes which can be considered for improving the manufacturability of these nodes.

Micro/nano-patterning of supported lipid bilayers (SLBs) has shown considerable potential for addressing fundamental biophysical questions about cell membrane behavior and the creation of a new generation of biosensors. Herein are presented several novel lithographic methods for the size-controlled patterning of SLBs from the microscale to the nanoscale. Using these methods, chemically distinct types of phospholipid bilayers and/or Escherichia Coli (E. Coli) membranes can be spatially addressed on a single microchip. These arrays can, in turn, be employed in the studies of multivalent ligand-receptor interactions, enzyme kinetics, SLBs size limitation, and membrane-associated species separation. The investigations performed in the Laboratory for Biological Surface Science include the following projects. Chapters II and III describe the creation of lab-on-a-chip based platforms by patterning SLBs in microfluidic devices, which were employed in high throughput binding assays for multivalent ligand-receptor interactions between cholera toxin B subunits (CTB) and ganglioside GM1. The studies on the effect of ligand density for multivalent CTB-GM1 interactions revealed that the CTB-GM1 binding weakened with increasing GM1 density. Such a result can be explained by the clustering of GM1 on the supported phospholipid membranes, which in turn inhibits the binding of CTB. Chapter IV characterizes the enzymatic activity of phosphatase tethered to SLBs in a microfluidic device. Higher turnover rate and catalytic efficiency were observed at low enzyme surface densities, ascribing to the low steric crowding hindrance and high enzyme fluidity, as well as the resulting improvement of substrate accessibility and affinity of enzyme catalytic sites. Chapter V presents sub-100 nm patterning of supported biomembranes by atomic force microscopy (AFM) based nanoshaving lithography. Stable SLBs formed by this method have a lower size limit of ~ 55 nm in width. This size limit stems from a balance between a favorable bilayer adhesion energy and an unfavorable bilayer edge energy. Finally, chapter VI demonstrates the electrophoretic separation of membrane-associated fluorophores in polymer-cushioned lipid bilayers. This electrophoretic method was applied to the separation of membrane proteins in E. Coli ghost membranes.

Novel micro patterning techniques have been developed for the patterning of volatile functional materials which cannot be conducted by conventional photolithography. First, in order to create micro patterns of volatile materials (such as bio-molecules and organic materials), micro-contact printing and shadow mask methods are investigated. A novel micro-contact printing technique was developed to generate micro patterns of volatile materials with variable size and density. A PDMS (Polydimethylsiloxane) stamp with 2-dimensional pyramidal tip arrays has been fabricated by anisotropic silicon etching and PDMS molding. The variable size of patterns was achieved by different external pressures on the PDMS stamp. A novel inking process was developed to enhance the uniformity and repeatability in micro-contact printing. The variable density of patterns could be obtained by alignment using x-y transitional stage and multiple stamping with a z-directional moving part. Second, for direct patterning of small molecule organic materials (e.g. pentacene), a novel shadow mask method has been developed with a simple and accurate alignment system. To make accurate dimensions of patterning windows, a silicon wafer was used for the shadow mask since a conventional semiconductor process gives a great advantage for accurate and repeatable fabrication processes. A sphere ball alignment system was developed for the accurate alignment between the shadow mask and the silicon substrate. In this alignment system, four matching pyramidal cavities were fabricated on each side of the shadow mask and silicon wafer substrate using an anisotropic silicon bulk etching. By placing four steel spheres in between the matching cavities, the self-alignment system could be demonstrated with 2-3um alignment accuracy in x-y directions. For OTFT (Organic thin film transistor) application, an organic semiconducting layer was directly deposited and patterned on the substrate using the developed shadow mask method. On the other hand, novel embedding techniques were developed for enabling conventional semiconductor processes including photolithography to be applied on the small substrate. The polymer embedding method was developed to provide an extended processing area as well as easy handling of the small substrate. As an application, post CMOS (Complementary metal-oxide-semiconductor) integration of a relatively large microstructure which might be even larger than the substrate was demonstrated on a VCO (Voltage-controlled oscillator) chip. In addition, micro patterning on the optical fiber was demonstrated by using a silicon wafer holder designed to surround and hold the optical fiber. The micro Fresnel lens could be successfully patterned and integrated on the optical fiber end.

In earlier research in our lab, the manipulation of microtubules on gold patterned silicon wafers was achieved by E-beam lithography, Poly (ethylene glycol) self assembled monolayers (PEG-SAMs) and electrophoresis. To develop a technique for delicate single microtubule manipulation, further studies need to be done on PEG-SAMs and electrophoresis. As a foundation of this goal, we examined the electric field in an aqueous solution between two planar electrodes and the compatibility of the antifouling property of PEG-SAMs with the electric field. For this purpose, the distribution of microbeads was analyzed using a Boltzmann distribution. The amount of adsorbed microtubules on a PEG-SAM was examined to test the compatibility of the antifouling property of a PEG-SAM with concomitant exposure to electric field. It is shown that the product of the electric field and the effective charge of the microbead does not have a linear relation with the applied electric potential but an exponentially increasing function with respect to the potential. The antifouling property of the PEG-SAM was not retained after an exposure to the electric field.

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 63-82).