In this study, we hypothesize that human acidic mammalian chitinase (AMCase) binds and is regulated by the epidermal growth factor receptor (EGFR), and that AMCase interacts with Galectin-3 (Gal-3) to mediate anti-apoptotic functions. We further hypothesize that asthma-associated polymorphisms of AMCase alter chitinase activity and modulate anti-apoptotic effects. We investigated the interactions between AMCase, Gal-3 and EGFR by establishing binding and co-expression in vitro; apoptotic effects were evaluated via Annexin V/Propidium Iodide staining. Molecular cloning was performed to generate single nucleotide polymorphisms (SNPs) of AMCase associated with asthma. Our data showed that co-expression of AMCase and EGFR induces chitinase activity; we found that AMCase and Gal-3 bind each other in vitro, and that they co-localize in the cytoplasm of cells. Co-transfection of AMCase and Gal-3 demonstrates greater anti-apoptotic effect than Gal-3 alone, while recombinant Gal-3 induces apoptosis, which is not blocked by incubation with recombinant AMCase. From these data, we conclude that AMCase is regulated by EGFR, and that AMCase and Gal-3 physically interact, however contrary to our hypothesis, the anti-apoptotic effects of AMCase are unlikely to be mediated by Gal-3. Further exploration of this pathway using SNP constructs generated in this study will shed light on the mechanism of AMCase in asthma.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 177-181).

The trend for designing of a titanium implant explored using different chemical compositions and crystallinity materials until people realized that the implant surface character was another important factor affecting the rate and extent of osseointegartion. Titanium received a macroporous titania surface layer by anodization, which contains open pores with average pore diameter around 5μm. An additional mesoporous titania top layer was created that followed the contour of the macropores and having 100–200 nm thickness and a pore diameter of 10 nm. Thus, a coherent laminar titania surface layer was obtained producing a hierarchical macro- and mesoporous surface. The interfacial bonding between the surface layers and the titanium matrix was characterized by a scratch test that confirmed a stable and strong bonding of the laminar titania surface layers upon titanium. The wettability to water and the effects on the osteosarcoma cell line (SaOS-2) proliferation and mineralization of the formed titania surface layers were studied systematically by cell culture and scanning electron microscopy (SEM). A synergistic role of the hierarchical macro- and mesoporosities was revealed in terms of enhancing cell adhesion, proliferation and mineralization, when compared with the titania surface with solo porosity scale topography. For the in vivo results of the evaluation of osseointegration, an argon ion beam polishing technique was applied to prepare the cross sections of implants feasible for the high resolution SEM investigation. The interfacial microstructure between newly formed bone and implants with four modified surfaces including the new hierarchical macro- and mesoporous implant surface retrieved after in vivo tests were characterized. By this approach it has become possible to directly observe early bone formation, the increase of bone density, and the evolution of bone structure. The two bone growth mechanisms, distant osteogenesis and contact osteogenesis, can also be distinguished. These direct observations give, at microscopic level, a better view of osseointegration and explain the functional mechanisms of various implant surfaces for osseointegration. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: In press. Paper 4: Manuscript.</p>

Estimation of the number of hidden states is challenging in hidden Markov models. Motivated by the analysis of a specific type of cell adhesion experiments, a new frame-work based on hidden Markov model and double penalized order selection is proposed. The order selection procedure is shown to be consistent in estimating the number of states. A modified Expectation-Maximization algorithm is introduced to efficiently estimate parameters in the model. Simulations show that the proposed framework outperforms existing methods. Applications of the proposed methodology to real data demonstrate the accuracy of estimating receptor-ligand bond lifetimes and waiting times which are essential in kinetic parameter estimation. The second part of the thesis is concerned with prediction of a deterministic response function y at some untried sites given values of y at a chosen set of design sites. The intended application is to computer experiments in which y is the output from a computer simulation and each design site represents a particular configuration of the input variables. A Bayesian version of the cubic spline method commonly used in numerical analysis is proposed, in which the random function that represents prior uncertainty about y is taken to be a specific stationary Gaussian process. An MCMC procedure is given for updating the prior given the observed y values. Simulation examples and a real data application are given to compare the performance of the Bayesian cubic spline with that of two existing methods.

The Signal Transducer and Activator of Transcription-3 (Stat3) is a transcription factor that is required for transformation by a number of oncogenes, while a constitutively active form of Stat3 alone is sufficient to induce neoplastic transformation. It was previously demonstrated that cell to cell adhesion causes a dramatic increase in the activity of Stat3 in both normal and tumour cells. This hinted for the first time at the possibility that the role of Stat3 may differ upon cellular confluence. To examine such a mechanism, it is important to evaluate the effect of Stat3 downregulation at different time-points relative to confluence. To examine this, two different approaches for Stat3 downregulation were used: (1) the introduction of high levels of peptidomimetics analogs, which block the Stat3-SH2 domain by using a technique of in situ electroporation. (2) Treatment with two platinum compounds that inhibit Stat3 binding to activated receptors and DNA. The results demonstrated that Stat3 downregulation in vSrc or TAg transformed mouse fibroblast cells or in breast carcinoma lines, induced apoptosis which was more pronounced post-confluence at the time of its peak activity. In contrast, in sparsely growing normal mouse fibroblasts, Stat3 inhibition induced merely a growth retardation. However, in densely growing normal fibroblasts, Stat3 inhibition induced apoptosis. At least in part, apoptosis induced by Stat3 inhibition was mediated by p53, as shown by the resistance to cell death by Stat3 downregulation in colon carcinoma cells, HCT116, where the p53 gene is ablated. Overall, our observations point to the possibility that constitutive activation of Stat3 may lead to tumourigenesis by downregulating wt-53 in cancers that do not have p53 mutations. As a result, targeting Stat3 in cancers with wt-p53 may be a promising therapeutic approach for restoring p53 function, thereby inducing p53-mediated apoptosis. Next, we examined the effect of constitutively activated Stat3 as an oncogene. Stat3C expression in rat F111 fibroblasts induced anchorage independence, but to a lower degree compared to other oncogenes, such as vSrc. Surprisingly Stat3C expression increased gap junction intercellular communication, despite the fact that other oncogenes such as vSrc or vRas effectively block gap junctions. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2009-04-26 01:09:21.654

<p>Diss. (sammanfattning) Umeå : Umeå universitet, 1996, härtill 6 uppsatser</p> / digitalisering@umu

Morphologic and metabolic change is a known property of the adult brain. A number of behavioural tasks alter local cerebral blood flow and glucose utilisation. The expression of the glucose transporter 1 (GLUT1), which allows the entry of glucose to the brain, also has been shown to change in response to long-lasting neuronal activation. However, little is known about the effect of acute neuronal activation on GLUT1 expression. Using immunohistochemistry and Western blot, we investigated cerebral GLUT1 expression and vasculature density in mice undergoing a 48-hour voluntary wheel running period. The results showed that the striatum was the main region where GLUT1 protein was up-regulated: There was a trend for GLUT1 expression and blood vessels density to be associated with the distance run during the experiment. These results indicate that short-term increased neuronal activation is associated with rapid changes in glucose transport and possibly vascular remodelling.

The local microenvironment of hematopoietic stem cells (HSCs) in the bone marrow -referred to as stem cell niche- is thought to regulate the balance of stem cell maintenance and differentiation by a complex interplay of extrinsic signals including spatial constraints, extracellular matrix (ECM) components and cell-cell interactions. To dissect the role of niche ECM components, a set of well-defined matrix biomolecular coatings including fibronectin, laminin, collagen IV, tropocollagen I, heparin, heparan sulphate, hyaluronic acid and co-fibrils of collagen I with heparin or hyaluronic acid were prepared and analyzed with respect to adhesive interactions of human CD133+ HSCs in vitro. ECM molecule dependent adhesion areas as well as fractions of adherent HSCs were assessed by reflection interference contrast microscopy and differential interference contrast microscopy. HSCs, so far mostly classified as suspension cells, exhibited intense adhesive interactions with fibronectin, laminin, collagen IV, heparin, heparan sulphate, and collagen I based co-fibrils. An integrin mediated adhesion on fibronectin and a L-selectin mediated adhesion on heparin pointed to specific interactions based on different adhesion mechanisms. As a consequence of HSC adhesion to molecules of the vascular and the endosteal regions, both regions were confirmed as possible stem cell niches and adhesive signals were suggested as potential regulators of stem cell fate. Furthermore, the impact of a spatially organized ECM on the HSC behavior was analyzed by single cell tracking. These studies required the development of engineered three-dimensional, ECM coated microcavities with the option for single cell tracking. A semi-automated cell-tracking tool was established to accelerate data access from time-lapse image sequences. From this analysis it was possible to reveal the genealogy, localization, morphology and migration of single HSCs over a time period of 4 days. A decreased cycling frequency was observed depending on the HSC localization in the spatially constraining microcavities. Besides the revealed impact of spatial constraints on HSC fate, the newly engineered ECM-coated microcavity setup and the semi-automated cell tracking tool provide new options to study the cell fate in engineered microenvironments at single cell level for other cell types ex vivo. / Die lokale Mikroumgebung von Blutstammzellen (BSZ) im Knochenmark, bezeichnet als Stammzellnische, reguliert das Gleichgewicht von Stammzellerhaltung und -differenzierung durch ein komplexes Zusammenspiel von extrinsischen Signalen wie räumliche Beschränkungen, Komponenten der extrazellulären Matrix (EZM) und Zell-Zell Wechselwirkungen. Um die Rolle der EZM-Komponenten zu analysieren, wurden definierte Beschichtungen von Fibronektin, Laminin, Kollagen IV, monomerem Kollagen I, Heparin, Heparan Sulphat, Hyaluronsäure und Co-Fibrillen aus Kollagen I und Heparin oder Hyaluronsäure hergestellt und in vitro bezüglich der adhäsiven Wechselwirkungen von humanen CD133+ BSZ untersucht. Die Adhäsionsflächen und der Anteil adhärenter Zellen wurden in Abhängigkeit von der EZM-Beschichtung mittels Reflexions- Interferenz-Kontrast-Mikroskopie und Differentieller Interferenz Kontrast Mikroskopie bestimmt. BSZ, bisher als Suspensionszellen definiert, zeigten intensive adhäsive Wechselwirkungen mit Fibronektin, Laminin, Kollagen IV, Heparin, Heparan Sulphat und den Co-Fibrillen. Eine Integrin abhängige Adhäsion auf Fibronektin und eine L-Selektin abhängige Adhäsion auf Heparin, wiesen auf spezifische Wechselwirkungen hin, die auf unterschiedlichen Mechanismen basieren. Aufgrund der Adhäsion von BSZ sowohl zu Molekülen der vaskulären als auch der endostealen Knochenmarkregion, wurden beide Bereiche als mögliche Stammzellnische bestätigt. Adhäsive Signale sind potentielle Regulatoren der Stammzellentwicklung. Im Weiteren wurde der Einfluss einer räumlich beschränkenden EZM auf das Verhalten der BSZ durch Einzelzellverfolgung untersucht. Diese Studien erforderten die Entwicklung von dreidimensionalen EZM-beschichteten Mikrokavitäten, die das Verfolgen einzelner Zellen ermöglichten. Es wurde ein halbautomatischer Algorithmus für die Zellverfolgung etabliert, um die Datengenerierung von den Zeitreihenaufnahmen zu beschleunigen. Die Analysen ermöglichten Aussagen über die Genealogie, Lokalisierung, Morphologie und Migration einzelner BSZ während einer Analysenzeit von 4 Tagen. Eine verringerte Zellteilungsaktivität wurde in Abhängigkeit von der BSZ Lokalisierung innerhalb der räumlich einschränkenden Mikrokavitäten festgestellt. Neben diesen Erkenntnissen bieten die entwickelten Mikrokavitäten und die etablierte Einzelzellverfolgung neue Möglichkeiten auch andere Zelltypen auf Einzelzellniveau ex vivo zu untersuchen.

A computational model of thrombus initiation and aggrandizement was proposed. The model separated the thrombotic process into three mechanisms, including shear enhanced diffusivity, platelet margination, and platelet adhesion. The model indicates that transport mechanisms may be the rate limiting condition of thrombus formation at physiological shear rates and that at higher shear rates; platelet binding becomes the rate limiting condition. Additionally a wall shear rate of 20000 s-1 and above should be considered as a new criterion for prophylactic treatment of an atherosclerotic lesion.

This dissertation aims to further the understanding of the complex communication that occurs as cells interact with topographical and chemical patterns on a biomaterial interface. The research accomplishes this through two aims fabricating cell substrate surface topography and chemical patterns independently using non-cleanroom approaches, and analyzing higher order cellular response to surface features. The work will impact biomaterial surface modification and fabrication which will apply to biomedical implanted devices, tissue engineering scaffolds, and biological analysis devices. The first aim seeks to apply non-traditional topographical and chemical patterning methods in order to create independent topographical and chemical patterns on cell culture substrates. Experiments use the resulting patterned substrates to quantify cellular alignment to surface topography and compare the relative influence of topographical and chemical patterns on cellular response. The combined patterning methods of imprint lithography and micro-contact printing result in a high-throughput technique applicable to a variety of materials and a range of feature sizes from nanoscale through microscale, thereby enabling future analysis of cell response to surface features. The second aim evaluates the impact of topographical and chemical features on cellular differentiation. Experiments use patterned topography overlaid with a characterized chemical model layer to evaluate the effects of topography on myoblast differentiation and alignment. Chemical patterns that independently control available cell spreading area and modulate cell-cell contact are used to investigate the impact of cell-cell contact on differentiation.