Diss. (sammanfattning) Stockholm : Karolinska institutet, 2009. / Härtill 4 uppsatser.

Thesis (M. Phil.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 49-59). Also available online.

Thesis (M. Phil.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 49-59). Also available in print.

Cigarette smoke's influence on cancer has primarily been a subject of epidemilogic and tumorigenic studies. There have been no proper investigations with interests focused on how cigarette smoke affects the cellular mechanics of metastasis. Gathering an understanding of how smoke influences metastatic invasion could be vital in regulating or possibly eliminatings cancer's ability to initiate new tumor growth sites. This project focuses on cigarette smoke's influence on cellular mechanics of endothelial cells, and the invasive potential of cancer against a fully active endothelium. It is already known that cigarette smoke has a carcinogenic effect, but it is hypothesized that the cigarette smoke causes the endothelium to exhibit pro-invasive characteristics. Cancer cells are often ignorant to extra-cellular stimuli. It is suspected that there will be a less pronounced degradation of cellular mechanics of cancerous cells than endothelial cells when exposed to similar concentrations of cigarette smoke.

published_or_final_version / Biological Sciences / Master / Master of Philosophy

Tendons, essential tissues that connect muscles to bones, are susceptible to rupture/degeneration due to their continuous use for enabling movement. Often surgical intervention is required to repair the tendon; relieving the pain and fixing the limited mobility that occurs from the damage. Unfortunately, post-surgery immobilization techniques required to restore tendon properties frequently lead to scar formation and reduced tendon range of motion. Our ultimate goal is to create an optimal tendon prosthetic that can stabilize the damaged muscle-bone connection and then be remodeled by resident cells from the surrounding tissues over time to ensure long-term function. To achieve this, we must first understand how cells respond to and interact with candidate replacement materials. The most abundant extracellular matrix (ECM) protein found in the body, collagen, is chosen as the replacement material because it makes up the majority of tendon dry mass and it can be remodeled by cell-based homeostatic processes. Previous studies found that Di-catechol nordihydroguaiaretic acid (NDGA) cross-linked fibers have greater mechanical strength than native tendons; and for this reason this biomaterial could be used for tendon replacement. This work focuses on investigating the behavior of fibroblasts on NDGA cross-linked and uncross-linked collagen samples to determine if cross-linking disrupts the cell binding sites affecting cell spreading, attachment, and migration. The in-vitro platform was designed by plasma treating 25 mm diameter cover slips that were exposed to 3-aminopropyl-trimetoxysilane/toluene and glutaraldehyde/ethanol solutions. The collagen solution was then dispensed onto the glutaraldehyde-coated cover slip and incubated for fibrillar collagen matrix formation. The collagen matrices were submerged in NDGA cross-linking solution for 24 hours to ensure the surface was completely cross-linked. Collagen films were made by allowing the uncross-linked gels to dry overnight before and after NDGA treatment, resulting in a more compacted structure. A spinning disk device was employed to quantify the ability of cells to remain attached to the collagen samples when exposed to hydrodynamic forces. To avoid any cell-cell interaction and focus on cell-surface interactions, 50-100 cells/mm2 were seeded carefully on each sample. Temporal studies demonstrated that cell adhesion strength and spreading area reached steady-state by 4 hr. Adhesion and spreading studies along with migration experiments demonstrated that NDGA treatment affects cellular behavior on films, partially reducing adhesion strength, migration, and spreading area. However, on the cross-linked gels which are less dense, the only change in cell behavior observed was in migration speed. We hypothesize that these differences are due to the collapsing of the collagen films. This compaction suggests a less open organization and could be allowing the collagen fibers to form more inter-chain bonds as well as bonds with the small NDGA cross-linker; while NDGA treatment of the fully hydrated gels may rely more on NDGA polymerization to span the greater distance between collagen fibrils. From these results, we can determine that the chemical/physical masking of the adhesion sites by NDGA on collagen films affects cellular behavior more than the masking that occurs in the cross-linked gels. Although this study shows an effect in cell behavior on the cross-linked films, it also demonstrates that cells can adhere and migrate to this NDGA biomaterial supporting the idea that this biomaterial can be utilized for tendon replacement.

Motivated by the need to design minimally-invasive treatments for wide-necked cerebral aneurysms, we used computational modeling to assess aneurysm hemodynamics, examined in vitro cellular responses arising from mechanical and chemical stresses, and designed novel materials that cooperatively adhere to the endothelium. We first hypothesized that because aneurysm geometry plays an important role in hemodynamics, changes in flow patterns may affect the shear stress experienced on the aneurysm wall. We defined flow regimes based on aneurysm hemodynamic and geometric parameters, which may correlate with aneurysm rupture. Because of the direct contact between endothelial cells (ECs) and blood flow, we then evaluated how changes in hemodynamics and inflammatory cytokines affect the expression of cell adhesion molecules (CAMs) and matrix remodeling factors on ECs. We subsequently designed biomaterials that complement the dynamic EC surface and have the ability to conform to any geometry through in situ crosslinking. Antibody-conjugated hydrogels facilitated synergistic EC adhesion using cooperativity as an adhesion strategy. We optimized the presentation of antibodies to inflammatory CAMs on polyvinyl alcohol (PVA) and alginate hydrogels to achieve strong adhesion to inflamed ECs. We synthesized photocrosslinkable, aminated PVA hydrogels and determined the effect of substrate stiffness on cell adhesion. We also evaluated the effects of antibody presentation on cell adhesion strength and dynamics using alginate hydrogels. Taken together, the results of this work may be used to design hydrogels for vascular remodeling applications under shear stress, including embolic agents for cerebral aneurysms. / Engineering and Applied Sciences

Skeletal muscle (SkM) possesses an impressive ability to regenerate in response to injury or chronic disease. This regenerative capacity is attributed to its resident mononuclear myogenic progenitors. Previous studies have identified several types of myogenic progenitors within SkM, some of which are isolated by fluorescence activated cell sorting (FACS) using cell surface markers. Studies in our laboratory have identified melanoma cell adhesion molecule (MCAM) as a cell surface marker expressed by myogenic progenitors in human fetal SkM. However, the relationship between MCAM expression and the degree of myogenic commitment of distinct MCAM+ populations has not been elucidated. In the present study, subpopulations of MCAM+ cells were purified by FACS on the basis of Hoechst 33342 dye uptake. Specifically, MCAM+ side population (SP) was isolated by Hoechst exclusion and MCAM+ main population (MP) on Hoechst incorporation. Sorted populations were first optimized for growth in vitro since SkM SP cells are difficult to maintain in culture. In particular, Invitrogen’s StemPro® MSC SFM medium was found to support propagation of human fetal SkM SP cells with minimal differentiation. Following this optimization, sorted populations were assessed for expression of myogenic markers before and after propagation and then for fusion potential in vitro and engraftment potential in vivo. The MCAM+ subpopulations were found to express myogenic markers to a significantly greater extent than MCAM- subpopulations. Furthermore, the MCAM+ subpopulations fused robustly into myotubes in vitro whereas the MCAM- subpopulations did not. Interestingly, the MCAM+ SP population exhibited the highest fusion potential in vitro and was the only MCAM+ subpopulation to engraft into dystrophic muscle in vivo following propagation. These results indicate that MCAM is associated with myogenicity and can be used to prospectively isolate a pure myogenic fraction from human fetal SkM tissue. Moreover, the MCAM+ SP retain its myogenic potential to a greater extent than MCAM+ MP after propagation. This suggests that the MCAM+ SP fraction contains a higher percentage of early myogenic progenitors compared to the MCAM+ MP fraction. Additional studies on MCAM-expressing populations in human fetal SkM may elucidate a potent population for use in cell-based therapeutic strategies for treating muscle diseases.

Huntington's disease (HD) is a rare, dominantly inherited neurodegenerative disorder characterized by progressive chorea, emotional and behavioral disturbances, and cognitive decline. The single, causative mutation is an expanded trinucleotide repeat of cytosine, adenosine, and guanine (CAG) of more than 37 residues in the HD gene (currently referred to as HTT). Genetic evidence suggests that the CAG repeat expansion results in a gain of huntingtin function. While huntingtin and its numerous interactors have been implicated in a variety of essential cellular processes, the role of the full-length, endogenous protein remains unclear. Multiple studies have implicated huntingtin in processes related to cytoskeletal structure and dynamics in HD patients and model organisms. However, alterations in cellular dynamics related to the cytoskeleton — including cell adhesion — have not been characterized in a comprehensive, rigorous manner. Using Mus musculus genetic models of the HD mutation and/or deficiency and a Dictyostelium discoideum genetic deficiency model, I have undertaken an investigation of evolutionarily conserved huntingtin function in the cytoskeleton and cell adhesion. The results of these studies support a role for huntingtin in cell-cell and cell-substrate adhesion, as well as maintaining actin cytoskeletal structure. Furthermore, my thesis research sets the stage for future work to elucidate the molecular mechanism by which huntingtin is acting and determine the effect of the CAG repeat expansion on huntingtin function. Evolutionary conservation affords an invaluable tool to identify crucial function(s) of the huntingtin molecule and the effect of the pathogenic HD mutation on function, enabling therapeutic development while providing novel insights into cytoskeletal biology and cell adhesion.

No description available.