In the Xenopus embryo, cell rearrangements during early development require the dynamic modulation of adhesion. Cells primarily use the integrin family of transmembrane receptors for attachment to and interpretation of the extracellular environment. While acting as adhesion receptors, integrins also have bidirectional signalling properties essential for driving cellular movements. The regulation of integrin activity is thought to stem from cytoplasmic assemblies of constitutively expressed molecules. PINCH (Particularly Interesting New cysteine-histidine rich protein), an adapter protein, is part of an IPP complex that has emerged as a key signalling scaffold indispensable for integrin function in vitro. As such, I tested the hypothesis that PINCH regulates integrin function in the Xenopus embryo. Xenopus PINCH was successfully cloned using RT-PCR. The predicted amino acid sequence of PINCH shares a 98% similarity with mammalian orthologs, and comprises of five highly conserved LIM domains. PINCH mRNA and protein are ubiquitously expressed throughout embryogenesis. In situ hybridization indicates that PINCH mRNA is expressed in the blastocoel roof and the pre-involution mesoderm. The localization and temporal expression of PINCH suggests a role in mediating cell adhesive events during gastrulation. A functional approach was used to examine the role of PINCH during gastrulation. I used site-directed mutagenesis to generate non-functional LIM1 (LIM1mut) and LIM4 (LIM4mut) domains that have been proposed to bind ILK and Grb4 respectively. Over-expression of PINCH leads to a delay in blastopore closures, while the expression of both LIM1mut and LIM4mut relieve this inhibition at lower concentrations. Further analysis indicates that PINCH, LIM1mut, and LIM4mut inhibit FN matrix assembly independent of integrin adhesion. Contradictory to in vitro studies, co-immunoprecipitation analysis indicates that endogenous PINCH does not bind ILK, confirming an integrin-independent role during gastrulation. Furthermore, in the embryo PINCH is found at cell boundaries but does not appear to directly modulate cadherin adhesion. As such this thesis provides evidence that PINCH regulates cell intercalation movements independent of integrin and cadherin receptors and raises the possibility that the LIM4 domain is involved in PINCH regulation of cell adhesion during early development.

<p>The number one cause of death in the industrialized world, atherosclerosis, can be treated through a variety of methods: angioplasty, stenting, vein graft bypass, synthetic grafts, and maybe one day tissue engineering vessels (TEBVs). The long term goal that motivated this research is the delivery of umbilical cord blood derived endothelial progenitor cells (CB-EPCs) to damaged arteries and possibly reducing the rate of re-occlusion by re-establishing a healthy, functional, intact endothelium. The proposed research tested the following hypotheses: (1) Mild trypsinization methods produces strong endothelial cell (EC) adhesion strength, (2) CB-EPCs are functionally similar to native ECs (specifically human aortic endothelial cells (HAECs)) and exhibit similar anti-thrombotic and anti-inflammatory behavior compared to HAECs, (3) CB-EPCs are capable of adhering to smooth muscle cells (SMCs) and extracellular matrix (ECM) proteins under flow conditions, (4) CB-EPCs can be used to prevent thrombosis in mice that have undergone vein bypass grafts through re-endothelialization of the vessel, and (5) CB-EPCs are capable of proliferating under flow conditions. In order to produce supraphysiological adhesion strengths of HAECs or CB-EPCs, the cells must be detached using 0.025% trypsin for 5 minutes prior to adhesion to adsorbed ECM proteins or SMCs. CB-EPCs have a high proliferation rate and express similar levels of important anti-thrombotic genes and inflammatory proteins compared to HAECs. CB-EPCs and HAECs produce similar levels of nitric oxide and alignment in the direction of flow when exposed to laminar shear stress for at least 24 hours. CB-EPCs are capable of adhering to many different substrates under flow conditions. The adhesion of CB-EPCs with response to shear stress appears to be biphasic and increases with shear stress up to 0.75 dyn/cm2 and then decreases above this value. CB-EPC adhesion is much greater than HAECs and EPCs isolated from peripheral blood (PB-EPCs) of healthy individuals, which can be related to their higher expression level of adhesion integrin &#945;5&#946;1 and their smaller size. When seeded onto FN coated plastic, CB-EPCs proliferated under flow conditions and had a much shorter doubling time than PB-EPCs and HAECs. Proliferation of CB-EPCs and HAECs on SMCs was limited. Further, Cb-EPCs formed network-like structures except when growth factors were removed and a shear stress of at least 5 dyne/cm2 was applied. To assess whether CP-EPCs could promote vessel repair in vivo, human CB-EPCs were injected into SCID mice that received a carotid interpositional vein grafts, resulting in 100% patency. In contrast, only 2 of the 8 saline injected mice had a patent vein graft 2 weeks post surgery. We found that CB-EPC injected mice had roughly 55% endothelialization compared to less than 20% for the patent saline controls, with CB-EPCs making up approximately 33% of this coverage. These results suggest that CB-EPCs could be used as a therapeutic method to prevent vessel re-occlusion in patients undergoing treatment for atherosclerosis.</p> / Dissertation

PVA hydrogels are potential biomaterials for various tissue-engineering applications. PVA hydrogels are relevant to vascular graft development due to their excellent biocompatibility and the capability to possess a wide range of mechanical properties based on compositional and processing parameters. This thesis aims to characterize some PVA hydrogels mechanically, biologically, and physically. A constitutive formulation is used for mechanical characterization, which allows for analysis of any possible stress-strain configuration applied to the material. A bovine aortic endothelial cell adhesion study under physiologic blood flow conditions comprises the biologic characterization, which gives insight into how human endothelial cells might interact with PVA hydrogels in a vascular graft application. A high-resolution SEM study is used to physically characterize the material, which furthers the understanding of the reactions of this material in vivo. These characterizations of PVA hydrogels will aid in the development of tissue-engineered products, in particular, the potential use as a vascular grafting biomaterial.

Cell adhesion to the extracellular matrix (ECM) provides tissue structure and integrity as well as triggers signals that regulate complex biological processes such as cell cycle progression and tissue-specific cell differentiation. Hence, cell adhesion is critical to numerous physiological and pathological processes, including embryonic development, cancer metastasis, and wound healing, as well as biotechnological applications, such as host responses to implanted devices and integration of tissue-engineered constructs. During the adhesion process, integrin surface receptors bind ECM proteins, cluster, and associate with the actin cytoskeleton. Subsequent strengthening of the integrin/actin cytoskeleton interaction occurs via complexes of proteins known as focal adhesions. Due to the close association between biochemical and biophysical processes within adhesion complexes, mechanical analyses can provide important new insights into structure/function relationships involved in regulating the adhesion process. The objective of this project was to investigate the role of the protein tyrosine kinase FAK in cell adhesion strengthening. Our central hypothesis was that FAK regulates adhesion strengthening by modulating interactions between integrins and FA structural components. Using a novel combination of genetically engineered cells to control the interactions of FAK, a spinning disk adhesion assay with micropatterned substrates to obtain reproducible and sensitive measurements of adhesion strength, and quantitative biochemical assays for analyzing changes in adhesive complexes, we demonstrate that FAK modulates adhesion strengthening via two distinct mechanisms: (1) FAK expression results in elevated integrin activation leading to regulation of strengthening rate and (2) FAK regulates steady-state adhesion strength via vinculin recruitment to focal adhesions. We also show that the autophosphorylation and catalytic sites of FAK are critical to this regulation of adhesion strengthening. This work is significant because it both identifies functional mechanisms of FAK and provides the first evidence that focal adhesion signaling regulates the adhesion strengthening process. Furthermore, this research demonstrates that the dependency of migration on adhesion strength is highly complex and establishes a need for adhesion strengthening metrics in analyzing the functional mechanisms of molecules within adhesion complexes.

Atomic force microscopy is one of the powerful instruments used to explore the mechanical properties of nanoscale materials. It not only can produce high-resolution images and surface mechanical properties, but also can make use of its probe for surface etching. In this study, we first use atomic force microscopy to measure the Adhesion Map of insulin amyloid fibers, then conduct mechanical lithography on the surface with the probe. In the end, we discuss the effect on insulin amyloid fibrils due to exert different forces and different speeds with the probe. According to Nanoindentation theory and Hertzian model, we can derive the Young's modulus of insulin amyloid fibrils from force-indentation relations. Then we cut the Insulin amyloid fibers with probe. The results showed that when we applied 3.23 nN force by the probe, the insulin amyloid fibers began to break. When we applied 7.07 nN force, insulin amyloid fibers are cut off easily. Therefore, we can bite off insulin amyloid fibers of different lengths and sections, and arrange in the desired pattern by atomic force microscope.

Nature encourages diversity in life forms (morphologies). The study of morphogenesis deals with understanding those processes that arise during the embryonic development of an organism. These processes control the organized spatial distribution of cells, which in turn gives rise to the characteristic form for the organism. Morphogenesis is a multi-scale modeling problem that can be studied at the molecular, cellular, and tissue levels. Here, we study the problem of morphogenesis at the cellular level by introducing an integrated biomechanical model of cells embedded in the extracellular matrix. The fundamental aspects of mechanobiology essential for studying morphogenesis at the cellular level are the cytoskeleton, extracellular matrix (ECM), and cell adhesion. Cells are modeled using tensegrity architecture. Our simulations demonstrate cellular events, such as differentiation, migration, and division using an extended tensegrity architecture that supports dynamic polymerization of the micro-filaments of the cell. Thus, our simulations add further support to the cellular tensegrity model. Viscoelastic behavior of extracellular matrix is modeled by extending one-dimensional mechanical models (by Maxwell and by Voigt) to three dimensions using finite element methods. The cell adhesion is modeled as a general Velcro-type model. We integrated the mechanics and dynamics of cell, ECM, and cell adhesion with a geometric model to create an integrated biomechanical model. In addition, the thesis discusses various computational issues, including generating the finite element mesh, mesh refinement, re-meshing, and solution mapping. As is known from a molecular level perspective, the genetic regulatory network of the organism controls this spatial distribution of cells along with some environmental factors modulating the process. The integrated biomechanical model presented here, besides generating interesting morphologies, can serve as a mesoscopic-scale platform upon which future work can correlate with the underlying genetic network.

High-resolution and low-cost fabrication techniques are essential for nanotechnology to overcome the commercialization barrier to benefit our society. Since its inception nanoimprint has become the ideal technology to fabricate dense sub-micron structures over large areas with low cost, which are important to many applications such as high-density storage disks and diffractive optical devices. The decade-long development in nanoimprint equipment has reached a point where large-scale manufacturing of high-density nanostructures are possible. However, there are a few remaining issues that need to be studied before the advent of commercial application of nanoimprint. In this work we look at a pressing issue, long-term stability of the mold surfactant coating. It is important to understand the details of the surfactant wear during nanoimprint in order to limit defect density to a tolerable threshold in a high-volume manufacturing process. To study this we went through a nanoimprinting procedure and measured chemical and physical alterations in the coating. The surfactant wear information also helps to optimize the time interval for surfactant recoating to keep the fabrication throughput as high as possible. In this paper we characterize the stability of two commonly used surfactants as well as prescribe a new technique for mold anti-adhesion. Through this work we see that FDTS and OTS undergo significant degradation in air and gradual degradation by chain scission is observed during the nanoimprint procedure. It is also noted that an embedded anti-adhesion layer is effective for mold releasing.

The Plastic Ball Grid Array (PBGA) package and flip-chip technology have been widely used in the microelectronics industry. However, due to the effect of hygroscopic and thermal stresses, the reliability is still of concern during manufacturing and operation, especially for the thermal-mechanical behavior of its corresponding interfaces. Influences of the storage conditions and reflow parameters on the warpage of the PBGA package are investigated in this study first. As the results, the warpage reflected the interaction of the extent of moisture absorption and the change in reflow parameters significantly. Furthermore, a critical relative moisture absorption between 0.25% and 0.30% is found for a considerable warpage response. Next, this study presents an experimental investigation of the adhesion strength of epoxy-based encapsulant material to solder mask coated FR-4 substrate under thermal cycling. Effects of the number of thermal cycles on the interfacial strength are investigated by using button shear test. The relationship between the interfacial strength and thickness of solder mask is also examined. Moreover, to characterize the degradation and fracture behavior, the morphologies of fractured surfaces of the test specimens are analyzed by scanning electron microscopy. The results of this experiment show that the interfacial strength of the epoxy-based encapsulant/solder mask/substrate joint is apparently reduced by thermal fatigue. And, the test specimen with larger solder mask thickness has higher interfacial strength. Finally, the single-lap joint test, nonlinear finite element analysis and Moir&#x00E9; interferometry are employed to obtain strain/stress distributions on the interface of solder mask and substrate. The effects of solder mask thickness and overlap length are then determined, separately. The results of this study can afford important information for characterizing the features of moisture absorption, warpage and interfacial adhesion of PBGA packages. Furthermore, it can be helpful to identify improvements required in reliability of the package design.

The effects of work¡¦s surface wavelength on the ultimate surface roughness are considered in this study. Both the experimental and theoretical studies will be done in this study. In the experimental study, the relationships between ultimate surface roughness and various operating parameters will be examined. In the theoretical study, a mathematical model relating the machining rate and various machining parameters are proposed. In the mathematical model, qualitative and quantitative properties of machining rate under various surface geometric condition are obtained by the aid of computer simulation. For the experimental study, a series of experiments will be done to investigate the effects of various factors on the ultimate surface roughness of different work¡¦s surface wavelength.The factors may include the the tool speed, the tool¡¦s surface irregularity, and the particle density of slurry. The comouter simulation indicates that the removal rate is non-linear proportional to tool speed and normal load. Besides, results also showed that the difference of removal rate between peak and valley of surface profile always decreases as the work¡¦s surface wavelength increases. The experimental study confirmed that the relationship between ultimate roughness and wavelength does exist in the specific range of work¡¦s surface wavelength . The model appears to be consistent with currently available experimental data.

Thesis (Ph. D.)--University of Chicago, Dept. of Chemistry, June 2001. / Includes bibliographical references. Also available on the Internet.