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Mechanics of Fibroblast Migration: a DissertationMunevar, Steven 09 May 2003 (has links)
Cell migration involves complex mechanical interactions between cells or between cells and the underlying substrate. Using a newly developed technique, "traction force microscopy", I have been able to visualize the dynamic characteristics of mechanical forces exerted by migrating fibroblasts such as magnitude, direction, and shear. For NIH 3T3 fibroblasts, I found that the lamellipodium provides nearly all of the force necessary for cell migration. A high shear zone separates the lamellipodium from the remainder of the cell body, suggesting that they are mechanically distinct entities. The timing of the tractions at the leading edge, as well as the spatial distribution, bears no apparent relationship to concurrent local protrusive activities, yet changes in traction force patterns often precede changes in migration direction. In H-ras transformed cells I found isolated regions of weak, transient traction forces in pseudopods all along the cell that appeared to act against one another. The resulting shear pattern suggested that there were multiple disorganized mechanical domains. These results support a frontal towing model for cell migration where the dynamic traction forces at the leading edge served to actively pull the cell body forward. In H-ras transformed cells, the weak poorly coordinated traction forces coupled with weak cell substrate-adhesions were likely responsible for the abnormal motile behavior of these cells. To probe the mechanical interactions beneath various regions of migrating fibroblasts, a cell substrate inhibitor (GRGDTP peptide) was locally applied while imaging stress distribution on the substrate utilizing traction force microscopy. I found that both spontaneous and GRGDTP induced detachment of the trailing edge resulted in extensive cell shortening with no change in overall traction force magnitude or cell migration. Conversely, leading edge disruption resulted in a dramatic global loss of traction forces pnor to any significant cell shortening. These results suggested that fibroblasts transmit their contractile forces to the substrate through two distinct types of adhesions. Leading edge adhesions were unique in their ability to transmit active propulsive forces whereas trailing end adhesions created passive resistance during cell migration and readily redistributed their loads upon detachment. I have also investigated how fibroblasts regulate traction forces based on mechanical input. My results showed that stretching forces applied through the flexible substrate induced increases in both intracellular calcium concentration and traction forces in fibroblasts. Treatment with gadolinium, a well known stretch-activated ion channel inhibitor, was found to inhibit both traction forces and cell migration without inhibiting cellular spread morphology or protrusive activities. Gadolinium treatment also caused a pronounced decrease in vinculin and phosphotyrosine concentrations from focal adhesions. Local application of gadolinium to the trailing region had no detectable effect on overall traction forces or cell migration, whereas local application to the leading edge caused a global inhibition of traction forces and an inhibition of migration. These observations suggest that stretch activated entry of calcium ions in the frontal region serves to regulate the organization of focal adhesions and the output of mechanical forces. Together my experiments elucidate how fibroblasts exert mechanical forces to propel their movements, and how fibroblasts utilize mechanical input to regulate their movements.
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Dynamic Control of Hydrogel Properties via Enzymatic ReactionsMoore, Dustin M. 05 1900 (has links)
Indiana University-Purdue University Indianapolis (IUPUI) / Dynamic changes to the extracellular matrix (ECM) impact many cell fate pro-
cesses. The ECM can experience changes in sti ness as well as changes in composi-
tion in response to injury, development, and diseases. To better understand the role
that these dynamic processes have on the cells residing within the environment, re-
searchers have turned towards 4-dimensional (4D) hydrogel designs. These 4D hydro-
gels re-capitulate not only 3-dimensional (3D) matrix architectures, but also temporal
changes in the physicochemical properties. The goal of this thesis was to design a
unify chemistry (i.e., Sortase A (SrtA)-mediated transpeptidation) for dynamic tun-
ing hydrogel sti ness and the presence of bioactive ligands. The rst objective was
to establish a tunable and cytocompatible enzymatic scheme for softening cell-laden
hydrogels. Brie
y, the e ects of SrtA-mediated matrix cleavage were investigated us-
ing poly(ethylene glycol) (PEG)-peptide hydrogels crosslinked by SrtA-sensitive and
insensitive peptides. Initially, the e ects of various parameters with respect to cat-
alytic reactions of SrtA were characterized rheologically, including enzyme and sub-
strate concentrations, macromer content, peptide composition, and treatment time.
Gel moduli pre- and post-enzyme treatment were measured to verify SrtA-mediated
hydrogel softening. The cytocompatibility of SrtA-mediated gel softening system was
investigated using human mesenchymal stem cell (hMSC). Upon treatment with SrtA
and an oligoglycine substrate, encapsulated hMSCs exhibited extensive spreading in
comparison to those within statically sti matrices. The second objective was to es-
tablish a reversible ligand exchange system utilizing SrtA-mediated transpeptidation.
SrtA-sensitive pendant ligands were immobilized within PEG hydrogels, which were treated with SrtA and an oligoglycine substrate to a ord tunable removal of the pen-
dant ligand. Through measurement of the liberated pendant peptide concentration,
it was found that higher concentrations of SrtA or extending treatment times led
to higher ligand removal e ciency. Finally, the e ect of peptide ligand removal on
cell behaviors were evaluated using NIH 3T3 broblasts. Fibroblasts were culture
both on and within hydrogels containing SrtA-cleavable cell adhesion peptide. After
treatment, both conditions led to a decrease in broblast spreading in comparison
to non-treated gels. Overall, the utility of SrtA as versatile agent for controlling the
mechanical properties and the presence of biologically active components within a
hydrogel system was demonstrated. These systems could be further explored with natural-based materials to better mimic the physiological environment experienced
by cells.
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Molecular and Proteomic Analysis of Components Involved in Abscisic Acid (ABA) Signaling NetworkSong, Jie 13 December 2014 (has links)
Abscisic acid is an important plant hormone in the responses to biotic and abiotic stresses, which also regulates various growth and developmental processes in plants. Three major components-receptors (PYRs), the PP2C type phosphatases and the SnRK2 subtype kinases form a double negative regulatory system: PYR/PYL/RCARs inhibit the activity of PP2Cs while PP2Cs inhibit that of SnRK2s in ABA signaling pathway. The results of my studies showed that ABA would directly affect the interaction between SnRK2.2 and ABI1 in absence of PYRs. Furthermore, ABA can inhibit the catalytic activity of the SnRK2.2 kinase. These findings indicated that ABA may directly interact with SnRK2.2. Posttranslational modifications play a key role in signal transduction. Phosphorylation is the most important posttranscriptional modification in ABA signal transduction. To dissect new components in ABA signaling network in plants, proteomics studies were carried out in Arabidopsis for identifying ABA- responsive phosphoproteins. Ten phosphoproteins, ATPB, ATPC1, FBA1, CTIMC, GGAT1, GAPC1, GAPC2, GAPA1 and ALDH11A3, were identified by 2-DE proteomic approach and LC-MS/MS analysis. These proteins are likely to be the potential phosphorylated targets of SnRK2s in ABA signaling network. Lysine acetylation (LysAc) also emerges as one of the important posttranslational modifications for protein regulation in plants. Eleven lysine acetylated proteins with altered acetylation in response to ABA were identified in Arabidopsis using proteomic approach. The increased LysAc of Rubisco and the decreased Rubisco activity by ABA treatment indicates the acetylation of Rubisco caused by ABA resulted in the inhibition of Rubisco activity. ABA has also been shown to exist and function in both lower animals and mammalians. The medical application of ABA has become a new area of investigation. To explore the role of protein phosphorylation in ABA-mediated function in mammalians, phosphoproteomic study was conducted in mouse 3T3-L1 cells. Ten phosphoproteins with significant changes in serine/threonine phosphorylation in response to ABA were identified. These results suggest these phosphoproteins are involved in ABA signaling network in mouse cells. The significance of the function of SFRS1, ANXA1 and Galectin-3 on human diseases indicated that ABA could be a potential treatment for some human diseases, such as cancer.
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Histone Deacetylase Inhibitors Trichostatin A (tsa) And Sulforaphane (sfn) Modulate Vitamin D Responsive Cyp24 Gene Expression in 3t3-l1 PreadipocytesAhn, Eunjee 01 January 2013 (has links) (PDF)
Vitamin D plays an important role in preserving healthy bones, and has additional roles in the body, including modulation of cell growth, differentiation, neuromuscular and immune function, and anti-inflammatory function. The vitamin D receptor (VDR) is a member of the nuclear hormone receptor superfamily and regulates transcription of vitamin D-dependent target genes, such as those for key proteins involved in calcium and phosphorus absorption and bone development. Histone acetylation weakens the association of histones with DNA, and increases the accessibility of transcriptional regulatory proteins to chromatin templates, thereby increasing transcriptional activity of gene expression. Histone deacetylases remove the acetyl groups and condense chromatin structure, thereby preventing transcription. TSA is a potent histone deacetylase inhibitor and can significantly enhance gene expression. Bioactive food component, sulforaphane (SFN) is found in cruciferous vegetables and is known to be a histone deacetylase inhibitor, leading to transcriptional activation of gene expression. The objective of this study is to demonstrate that the bioactive food components modulate vitamin D action in adipocytes. To investigate the effects of TSA and SFN on vitamin D response, 3T3L1 mouse preadipocytes were treated with the combination of various concentrations of 1,25(OH)2 vitamin D, TSA, and SFN. Upon harvesting cells, the amounts of 24-hydroxylase mRNA, marker of vitamin D response, were measured by semiquantitative reverse transcriptase-PCR analysis. The results showed that the cells treated with 1μM TSA increased 1,25(OH)2 vitamin D-induced CYP24 mRNA level nearly 3.5-fold (p < 0.05) at 1nM 1,25(OH)2 vitamin D and nearly 2.5-fold (p < 0.05) in 10 nM 1,25(OH)2 vitamin D, and the cells treated with 5μM SFN increased 1,25(OH)2 vitamin D-induced CYP24 mRNA level nearly 1.4-fold at 1nM 1,25(OH)2 vitamin D and nearly 1.2-fold at 10 nM 1,25(OH)2 vitamin D.
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Vitamin D Metabolites Inhibit Adipocyte Differentiation in 3t3-l1 PreadipocytesNatarajan, Radhika 01 January 2008 (has links) (PDF)
No description available.
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Nitric Oxide Synthase in Confined Environments: Detection and Quantification of Nitric Oxide Released From Cells and Modified Liposomes Using a Sensitive Metal Catalyst-PEDOT Modified Carbon Fiber ElectrodePerera, Reshani H. January 2009 (has links)
No description available.
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Matrigel alters the expression of genes related to adipogenesis and the production of extracellular matrix in 3T3-L1 cellsJosan, Chitmandeep January 2018 (has links)
Studying molecular mechanisms underlying adipocyte differentiation is imperative to understanding adipocyte function and its role in obesity. However, the majority of research exploring adipogenesis is conducted with cell lines cultured directly on tissue culture plastic. Culturing cells on plastic may result in altered proliferation and differentiation, and subsequent change in pharmacological response. The extracellular matrix (ECM) plays a critical role in adipocyte development and survival. It is suggested that cells in vitro express high levels of ECM proteins to compensate for lack of an ECM. Differentiating preadipocytes on a substrate representative of the mature adipocyte extracellular environment may provide a more physiological response to drugs and environmental chemicals. The purpose of this study was to investigate the impact of Matrigel on 3T3-L1 cell growth, differentiation, lipid accumulation and responsiveness to Rosiglitazone. Matrigel decreased 3T3-L1 cell proliferation, enhanced lipid accumulation, and increased expression of adipogenic and lipogenic markers, including PPARγ, C/EBPα, SREBP1c, FAS, LPL, FABP4 and PLIN1. This was accompanied by a decrease in gene expression of ECM proteins, including fibronectin, collagen 1, collagen 3, collagen 4, laminin and collagen 6 in 3T3-L1 cells on Matrigel. Finally, Matrigel enhanced the response of 3T3-L1 cells to Rosiglitazone, which is a known PPARγ agonist and significantly increases lipid accumulation in 3T3-L1 cells. Our results suggest that enhanced lipid accumulation in 3T3-L1 cells on Matrigel is associated with decreased expression of ECM genes. Future studies require investigation of the cell-to-ECM interaction to confirm these findings. This study proposes that the nature of the ECM for cultured adipocytes alters temporal lipid accumulation patterns and response to various drugs as compared to 3T3-L1 cells grown on tissue culture plastic. / Thesis / Master of Science (MSc)
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The Effect of Transforming Growth Factor Beta (TGF-β3) and Sanicle on Wound Healing.Beggs, Clive B., Denyer, Morgan C.T., Lemmerz, A., Sefat, Farshid, Wright, Colin W., Youseffi, Mansour 2010 March 1915 (has links)
No / There is evidence that both the herb Sanicle and the
cytokine TGF- β3 can be beneficial in enhancing wound repair.
In this study 3T3 fibroblast cells were cultured and the
confluent monolayers were wounded (scarred) using a
disposable plastic pipette. Various amounts of TGF-β3 (a
growth factor) and Sanicle extract were applied to the cell
monolayers. TGF-β3 was applied at concentrations of 50ng/ml,
5ng/ml, 500pg/ml, 50pg/ml and 5pg/ml to five different culture
flasks with one additional flask acting as control. Sanicle was
applied at concentrations of 100μg/ml, 10μg/ml, 1μg/ml,
100ng/ml and 10ng/ml with one additional flask as a control.
The cells were imaged over a period of 20 hours with or without
presence of TGF-β3 and Sanicle. The results indicated that
although there were no significant increases in the rate of wound
closure in relation to application of TGF-β3, there is an
indication that TGF-β3 may enhance model wound closure at
optimum working concentration between 5ng/ml and 50ng/ml.
However, the sanicle extract did not stimulate enhanced repair
of the model in vitro wound, but instead seemed to promote cell
death along the wound margin. These results indicate that
sanicle may be used in the care of wounds, but not as a growth
promoter, but because it acts as an antibiotic agent, and possibly
because it aids wound debridement.
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Cree traditional medicine and type 2 diabetes : an ethnobotanical and pharmacological studyLeduc, Charles January 2005 (has links)
Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.
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Investigations of in vitro test systems for the detection of Glucocorticoid-induced skin atrophy as a tool in drug discoverySchoepe, Stefanie 12 August 2009 (has links)
Topische Glukokortikoide (GCs) sind wirksam bei Therapie von entzündlichen Hauterkrankungen. Durch ihr Nebenwirkungspotential (z.B. Induktion von Hautatrophie) ist ihr Einsatz jedoch begrenzt. Für die Medikamentenentwicklung ist die Bestimmung des atrophogenen Potenzials neuer Verbindungen daher von großer Bedeutung. Derzeit stehen dafür keine prädiktiven in vitro Modelle zur Verfügung. Ziel dieser Arbeit war daher die Etablierung solcher Modelle. Es wurden kutane Zelltypen (3T3-Zellen, Rattenfibroblasten, HaCaT-Zellen, humane Keratinozyten [NHEK] und Fibroblasten) und Vollhautmodelle (CellSystems AST-2000 und Phenions FTSM) untersucht. Atrophie-Marker, die Proliferation, Kollagen-Metabolismus und Epidermisdicke betreffend, wurden auf mRNA-, Protein- bzw. zellulärer Ebene gemessen. Außerdem wurden mittels Genexpressionsanalysen von GC-behandelter Nagerhaut neue potenzielle Marker identifiziert, deren Regulation in vitro jedoch nicht bestätigt werden konnte. Nach Pilotexperimenten wurden 3 Modelle ausgewählt und für Evaluierungsexperimente mit Referenz-GCs behandelt: 1). MMP1, -2, -3 und -9 mRNA-Expression in NHEK, 2). COL1A1 und COL3A1 mRNA-Expression in 3T3-Zellen, 3.) Epidermisdicke, Kollagen- und MMP-Synthese in FTSM. Die Messparameter der 3 Modelle erwiesen sich als dosisabhängig reguliert und korrelierten mit dem atrophogenen Potenzial der GCs. Schließlich wurde die Prädiktabilität der 3 in vitro Modelle für die in vivo Situation im Nager analysiert. In allen 3 in vitro Systemen induzierte die Behandlung mit einem selektiven GC-Rezeptor-Agonisten weniger atrophogene Effekte als das Referenz-GC. Ähnliche Ergebnisse wurden auch in vivo im Rattenhautatrophie-Modell gefunden. Zusammenfassend wird eine Kaskade von 3 in vitro Modellen empfohlen, um das atrophogene Potential von GC-Rezeptor-Liganden zu bestimmen. Der tatsächliche prädiktive Wert für die klinische Situation sollte in weiteren Studien untersucht werden. / Topical glucocorticoids (GCs) are effective for the therapy of inflammatory skin diseases. However, their use is limited by their side effect potential, with skin atrophy being the most prominent one. Thus, determining the atrophogenic potential of novel compounds is of importance for drug development. Currently, there are no according predictive in vitro models available. The aim of this study was to establish such atrophy models. Rodent and human cutaneous cell types (3T3 cells, rat fibroblasts, HaCaT cells, human keratinocytes [NHEK] and fibroblasts) and human full-thickness skin equivalents (CellSystems AST-2000 and Phenions FTSM) were investigated. Atrophy markers related to proliferation, collagen metabolism and epidermal thickness were measured on mRNA, protein and cellular level, respectively. Additionally, by gene expression profiling of GC-treated rodent skin novel potential markers were identified, but subsequently not confirmed in vitro. After pilot studies 3 models were selected and treated with reference GCs for evaluation experiments: 1.) MMP1, -2, -3 and -9 mRNA expression in NHEK, 2.) COL1A1 and COL3A1 mRNA expression in 3T3 cells, 3.) epidermal thickness, collagen and MMP synthesis in FTSM. The read out parameters of all 3 test systems turned out to be regulated dose-dependently and correlated with the atrophogenic potential of the GCs. Finally, the predictability of the 3 recommended in vitro test system for the rodent in vivo situation was analyzed. In all 3 in vitro test systems, the treatment with a novel selective GC receptor agonist induced less atrophogenic effects than the reference GC clobetasol. Indeed, similar results were found in the hr/hr rat skin atrophy model. In summary, a cascade of 3 in vitro models is recommended to be applied for the characterization of the atrophogenicity of GC receptor ligands. Further experiments are necessary to eventually demonstrate the true predictability of these models for the clinical situation.
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