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In biomechano-transducing, cellular generated tension can be measured by soft substrates based on polymers but these techniques are limited either by spatial resolution or ability to detect localised cell traction forces (CTF) due to their non-linear viscous behaviour under shear rates. A newly developed cell traction force transducer system based on cholesteryl ester lyotropic liquid crystals (LCTFT) was developed to sense localised traction forces of human keratinocyte cell lines (HaCaTs), in which the length of the deformation line induced represents the intensity of the CTF exerted. The physical properties of the cholesteryl ester based lyotropic liquid crystals (LLC) were characterised by using polarising microscopy, rheology, atomic force microscopy (AFM) based nano-indentation, spherical indentation, and micro-tensile tests. The interactions of LLC with cells were studied by using cell viability studies, cytochemical treatments, widefield surface plasmon resonance (WSPR) microscopy and various immuno-staining techniques. The results show that LLC is thermally stable (0 - 50 oC) and linearly viscoelastic below 10 % shear strain at shear rates of < 1 s-1. AFM nano and spherical indentations show a good agreement on the Young¿s modulus of both determined at ~110 kPa which is close to the elastic modulus of the epidermis. The Poisson¿s ratio of LLC was determined at ~0.58 by using micro tensile tests. The biophysical interaction studies indicated that LLC is biocompatible and allowed cell attachment. Cell relaxation technique by cytochalasin-B treatment suggested that the attachment and contraction of cells on LLC was due to the contractile activity of actin cytoskeletons that are mediated by focal adhesions. The staining experiments showed that cells consistently expressed the same suites of integrins (¿2, ¿3, ¿5 and ¿1) and ECM proteins (collagen type IV, laminin and fibronectin) on both glass and LLC coated substrates. Interfacial interaction of cells with LLC observed via the staining of actin and vinculin, and WSPR imaging suggest the association of marginal actin filaments and focal adhesions in attaching HaCaT cells to the LLC. Linear static analysis applied in the Finite Element model of focal adhesion-LC confirmed the compressive force patterns induced by cells. By applying cell relaxation techniques and Hooke¿s theorem, the force-deformation relationships of the LLC were derived and used for direct quantification of CTF in culture. The sensitivity of the LCTFT was implied by a wide range of CTF (10 - 140 nN) measured at high resolutions (~2 ¿m). Nonetheless, a custom-built cell traction force measurement and mapping software (CTFM) was developed to map CTF of single cells. Reliability of the LCTFT was evaluated by using a known pharmacological active cytokine, TGF-¿1, in inducing contraction of human keratinocytes. This study inferred internal consistency and repeatability of the LCTFT in sensing contraction responses of HaCaT cells in a concentration dependent manner of TGF-¿1. The overall LCTFT and CTFM software had shown good potential for use in the study of contraction and migration of keratinocytes. / Malaysia Ministry of Higher Education

In biomechano-transducing, cellular generated tension can be measured by soft substrates based on polymers but these techniques are limited either by spatial resolution or ability to detect localised cell traction forces (CTF) due to their non-linear viscous behaviour under shear rates. A newly developed cell traction force transducer system based on cholesteryl ester lyotropic liquid crystals (LCTFT) was developed to sense localised traction forces of human keratinocyte cell lines (HaCaTs), in which the length of the deformation line induced represents the intensity of the CTF exerted. The physical properties of the cholesteryl ester based lyotropic liquid crystals (LLC) were characterised by using polarising microscopy, rheology, atomic force microscopy (AFM) based nano-indentation, spherical indentation, and micro-tensile tests. The interactions of LLC with cells were studied by using cell viability studies, cytochemical treatments, widefield surface plasmon resonance (WSPR) microscopy and various immuno-staining techniques. The results show that LLC is thermally stable (0-50 °C) and linearly viscoelastic below 10% shear strain at shear rates of < 1 s⁻¹. AFM nano and spherical indentations show a good agreement on the Young's modulus of both determined at ~110 kPa which is close to the elastic modulus of the epidermis. The Poisson's ratio of LLC was determined at ~0.58 by using micro tensile tests. The biophysical interaction studies indicated that LLC is biocompatible and allowed cell attachment. Cell relaxation technique by cytochalasin-B treatment suggested that the attachment and contraction of cells on LLC was due to the contractile activity of actin cytoskeletons that are mediated by focal adhesions. The staining experiments showed that cells consistently expressed the same suites of integrins (α2, α3, α5 and β1) and ECM proteins (collagen type IV, laminin and fibronectin) on both glass and LLC coated substrates. Interfacial interaction of cells with LLC observed via the staining of actin and vinculin, and WSPR imaging suggest the association of marginal actin filaments and focal adhesions in attaching HaCaT cells to the LLC. Linear static analysis applied in the Finite Element model of focal adhesion-LC confirmed the compressive force patterns induced by cells. By applying cell relaxation techniques and Hooke's theorem, the force-deformation relationships of the LLC were derived and used for direct quantification of CTF in culture. The sensitivity of the LCTFT was implied by a wide range of CTF (10 - 140 nN) measured at high resolutions (~2 μm). Nonetheless, a custom-built cell traction force measurement and mapping software (CTFM) was developed to map CTF of single cells. Reliability of the LCTFT was evaluated by using a known pharmacological active cytokine, TGF-β1, in inducing contraction of human keratinocytes. This study inferred internal consistency and repeatability of the LCTFT in sensing contraction responses of HaCaT cells in a concentration dependent manner of TGF-β1. The overall LCTFT and CTFM software had shown good potential for use in the study of contraction and migration of keratinocytes.

Formins, also known as formin homology (FH) proteins, are involved in a wide range of actin-mediated processes. The Diaphanous-related formin Daam1 (Dishevelled-associated activator of morphogenesis) interacts with the PDZ domain protein Dishevelled, and is required to establish planar cell polarity in Xenopus. Through a yeast two-hybrid screen, I characterized a PDZ-mediated interaction between the C-terminus of Daam1 and the PDZ domains 456 of GRIP1. In dissociated rat hippocampal cultures, Daam1 expression was seen throughout the soma and dendrites in a punctate pattern. Furthermore, co-staining with a synaptic marker suggests that Daam1 could be associated with post-synaptic specializations. Dendritic spines are enriched with actin filaments, and based on the subcellular localization of Daam1 and the evidence that formins are involved in regulating actin polymerization, I hypothesized that Daam1 might play a role in dendritic spine morphology. In order to investigate the functional roles for Daam1, viral vectors were developed using the Semliki-Forest defective viral vector to over-express the full-length Daam1 protein and a Daam1 lacking the PDZ-binding motif. The over-expression of the full-length Daam1 in organotypic hippocampal slices showed a punctate distribution throughout the dendritic shaft, with the occasional appearance in spines, resulting in an overall increase in dendritic spine length. This suggests that formins, such as Daam1, could potentially regulate spine morphology.

Cell adhesion is essential to cell motility and tissue integrity and is regulated by the Integrin family of transmembrane receptors. Integrin binds to ligand extracellularly and provide anchor to the intracellular cytoskeleton via adhesion scaffolding proteins. In order to link cell to the surrounding matrix Integrin needs to be activated. Intracellular activation signals induce perturbations in Integrin cytoplasmic domain that are translated into a conformational change in extracellular region for high affinity ligand binding. Integrin engagement by matrix, in turn, triggers the assembly of adhesion complexes. Such early adhesions promote cytoskeletal organization with subsequent contractile activity that exerts forces against initial Integrin-matrix adhesions. In response to force, Integrin strengthens the interaction with matrix through its clustering and successive recruitment of additional adhesion components. These bidirectional regulatory loops mediated by such interactions are largely dependent on the unique function of Integrin adhesion components. / We demonstrate a novel role for the PDZ/LIM domain protein Zasp as a core component of Integrin adhesions. Specifically, Zasp colocalizes with Integrins at focal adhesion in cultured cells and myotendinous junctions in Drosophila embryos. In both cases elimination of Zasp modifies Integrin function causing consequently defects in cell spreading and muscle attachment. Zasp supports Integrin adhesion to the extracellular matrix that is required to withstand tensile forces exerted during cell spreading and muscle contraction. Furthermore, we found that the distribution of Zasp in muscle Z-lines is essential to orchestrate the cross-linking of alpha-Actinin and Actin filaments. Disruption of Zasp leads to loss of muscle cytoarchitecture, pointing to a larger role for Zasp in sarcomere assembly. Finally, we demonstrate that Zasp, in addition to alpha-Actinin, physically interacts with the Integrin- and Actin-bound cytoskeletal protein Talin. / Collectively, our results point to a dual role for Zasp as a structural scaffold. First it regulates Integrin adhesion to the extracellular matrix by interacting with the head domain of Talin at the myotendinous junctions. Second, Zasp controls sarcomere assembly by tethering the presarcomeric alpha-Actinin component to the tail domain of Talin. Zasp finding as a crucial adhesion component provides further insights on the mechanism underlying Integrin-mediated adhesion.

Thesis (Ph.D. in Neuroscience) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 110-125). Online version available via ProQuest Digital Dissertations.

Exposure to external stimuli promotes a variety of cellular responses including changes in morphology, gene expression and cell division status. These responses are promoted by signaling pathways composed of modules that are conserved from lower to higher eukaryotes. In Saccharomyces cerevisiae response to the external stimuli provided by mating pheromone is governed by the pheromone response pathway. This pathway is composed of a G protein coupled receptor/heterotrimeric G protein (Gαβγ) module and a MAP kinase cascade. Activation of this pathway allows the heterotrimeric G protein βγ dimer (Gβγ) to recruit polarity proteins to promote changes in cell morphology and to activate signaling through the MAP kinase cascade. Here we investigate the regulation of these pheromone-induced responses. We first examine how an asymmetric polarization response is generated. Normally, a gradient of pheromone serves as a spatial cue for formation of a polarized mating projection, but cells can still polarize when pheromone is present uniformly. Here we show that an intact receptor/Gαβγ module is required for polarization in response to both a gradient and uniform concentration of pheromone. Further investigation into regulation of Gβγ by Gα revealed that the two interaction interfaces between Gα and Gβ have qualitatively different roles. Our results suggest that one interface controls signaling whereas the other governs coupling to the receptor. Overall our results indicate that communication between the receptor and Gαβγ is required for proper polarization. We then examine how G1 CDKs regulate MAP kinase signaling. Response to pheromone is restricted to the G1 stage of the cell cycle. Once cells commit to a round of division they become refractory to mating pheromone until that round of division is complete. One contributor to this specificity involves inhibition of signaling through the MAP kinase cascade by G1 CDKs, but it was not known how this occurs. Here, we show that the MAP kinase cascade scaffold Ste5 is the target of this inhibition. Cln/CDKs inhibit signaling by phosphorylating sites surrounding a small membrane-binding domain in Ste5, thereby disrupting the membrane localization of Ste5. Furthermore, we found that disrupting this regulation allows cells to arrest at an aberrant non-G1 position. Our findings define a mechanism and a physiological benefit for restricting pheromone-induced signaling to G1. This thesis describes findings related to generation of an asymmetric polarization response, heterotrimeric G protein function, and coordination of differentiation signaling with cell division status. Lessons learned here might be applicable to the regulation of polarization and differentiation responses in other systems as the signaling modules are conserved.

In cancer, aberrant activation of developmental signaling pathways such as the Hippo Pathway has been shown to drive proliferation and invasion of cancer cells. Therefore, understanding the normal function of the Hippo Pathway during embryonic development can provide critical insight into how aberrant activity contributes to tumorigenesis. This dissertation explores the role of the Hippo Pathway members YAP and TAZ in gastrointestinal (GI) development and tumorigenesis. I use mouse genetics to systematically dissect the roles of YAP/TAZ in the endoderm-derived gastrointestinal epithelia and mesoderm-derived gastrointestinal mesenchyme during mammalian development. In the GI epithelium, I demonstrate that YAP/TAZ are dispensable for development and homeostasis. However, YAP/TAZ are required for Wnt pathway-driven tumorigenesis. I find that YAP/TAZ are direct transcriptional targets of Wnt/TCF4 signaling. In the GI mesenchyme, I describe a previously unknown requirement for YAP/TAZ activity during mammalian GI development. YAP/TAZ are involved in normal GI mesenchymal differentiation and function as transcriptional co-repressors in a progenitor cell population. In this way, YAP/TAZ act as molecular gatekeepers prior to Hedgehog-mediated differentiation into smooth muscle cells. This work unveils a previously unknown requirement for Hippo pathway signaling in the mammalian GI tract and a novel mechanism wherein YAP/TAZ function as transcriptional co-repressors to maintain a mesenchymal progenitor cell population.

Adaptive immunity requires T cell responses to foreign pathogens to be counterbalanced with the need to limit collateral destruction of the host’s own tissues. Further, the presence of a substantial pool of lymphocytes capable of recognizing selfantigen in the periphery poses a threat to the maintenance of peripheral tolerance and prevention of autoimmunity. Regulatory T cells (Treg) that can suppress potentially self-reactive T cells are critical regulators of peripheral tolerance as well as initiation of immune responses. Treg cells employ several context-dependent mechanisms to establish regulation. In this thesis, we describe two distinct pathways of regulation used by Treg cells involving negative costimulation by CTLA-4 and immunomodulation by the morphogen, TGFβ. CTLA-4 is a co-inhibitory receptor on T cells essential for maintaining T cell homeostasis and tolerance to self. CTLA-4 expression is induced in conventional T cells following activation, whereas it is constitutively expressed in regulatory FOXP3+CD4+ regulatory T cells. Mice lacking CTLA-4 develop an early onset, fatal breakdown in T cell tolerance. Whether this autoimmune disease occurs because of the loss of CTLA-4 function in regulatory T cells, conventional T cells, or both, is not known. We present evidence here that in addition to a critical CTLA-4 function in regulatory T cells, CTLA-4 in conventional T cells is also necessary for controlling the consequences of abnormal T cell activation. CTLA-4 expression in activated conventional T cells only in vivois unable to compensate for the impaired function of CTLA-4-less regulatory T cells that results in systemic lymphoproliferation, but it can prevent the aberrantly activated T cells from infiltrating and fatally damaging non-lymphoid tissues. These results demonstrate that CTLA-4 has a dual function in maintaining T cell homeostasis: CTLA-4 in regulatory T cells inhibits inappropriate naïve T cell activation and CTLA-4 in conventional T cells can prevent the harmful accumulation of inappropriately activated pathogenic T cells in vital organs. In addition, we have identified Disabled-2 (Dab2), a TGFβ signaling intermediate, as a FOXP3 target gene that is expressed exclusively in Treg cells and is critical for in vitro and in vivo regulation by Treg cells. During T cell development, DAB2 is also expressed in a Foxp3-independent manner in thymic precursor cells, and acts as a sensor of TGFβ signals that is required for programming normal TGFβ responsiveness in T cell progenies. Naïve CD4+ T cells that differentiate from Dab2-deficient precursors favor Th17 cell generation at the expense of FOXP3+ Treg cells as a result of altered sensitivity to TGFβ. Importantly, retinoic acid can restore TGFβ signaling capacity of naïve CD4+ T cells generated from Dab2-deficient precursors, emphasizing the cooperative nature of retinoic acid and TGFβ signaling pathways in promoting Treg cell development and maintenance.

No description available.