DEPARTAMENTO DE CIÊNCIAS DA VIDA ... - Estudo Geral

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7 transduced to affect cellular signaling, gene expression and cell function, which are crucial to developmental programming (Lee et al., 2006; Somogyi and Rorth, 2004; Farge, 2003). (Ingber et al., 1986a; Ingber and Folkman, 1989b). It was shown that changes in the balance of mechanical forces between integrins and cytoskeleton that accompany cells spreading and drive cytoskeletal restructuring control downstream mitogenic signaling cascades and thereby control the cellular response to other external stimuli. So mechanical forces are equally important biological regulators as growth factors and ECM are (Huang et al., 1998). It has long been noted that the differentiation of stem cells into multiple lineages is accompanied by dramatic changes in cell morphologies, and the demonstration that mechanical cues influence lineage commitment of stem cells (McBeath et al., 2004). So, inhibition of cell-generated forces was showed to alter many basic cellular functions, such as proliferation, differentiation, tissue organization and migration (Huang et al., 1998; McBeath et al., 2004; Sordella et al., 2003; Krieg et al., 2008; Lo et al., 2004). By modulating the stress that is generated by these cellular forces altering the mechanical stiffness of the substrate it is possible to achieve a similar response to the one caused by directly altering cellular contractility (Pelham and Wang, 1997; Paszek and Weaver, 2004; Engler et al., 2006). Stiffness is the extent to which a substrate resists deformation. Substrate stiffness itself can alter numerous cellular functions including migration, proliferation and differentiation (Li et al., 2007; Peyton and Putnam, 2005; Leach et al., 2007; Lo et al., 2000).
Figure 1. Mechanotransduction in a Cell-ECM Unit: Center image – A cell connected to another cell and to the ECM. Center image (A) and (D)- show where mechanotransduction in the cell-ECM unit occurs. The “blue lines” represent actomyosin filaments, “green lines” embody intermediate filaments and the “red lines” correspond to microtubules in all panels. Integrins are represented by the “blue structures” linking the cell with the ECM (D); Center “nucl.” – nucleus; (A) -Mechanotransduction at adherens junctions; (A), LEFT Shows different cell-cell junctions –“Tight” is for tight junctions. “GAP” is for Gap junctions. “Desm” is for desmosomes. “AJ” is for Adherens junctions.(A), RIGHT: Shows the Molecular structure of an AJ: E-cad: E-Cadherin, a: Alpha Catenin, b: Beta Catenin, p120: p120 Catenin, v: Vinculin; (B) -Mechanoreceptors at the cell membrane. Deformation of the plasma membrane by the fluid flow or stretching, leads to activation of ion channels resulting in an ions influx. Furthermore fluid flow directly impacts glycocalyx and cilia movement which triggers diverse downstream signaling cascades. Mechanical forces also mediate growth factor receptor (GR) clustering and endocytosis, and thus affect GR signal transduction as well; (C) - Mechanotransduction at the nucleus. Intermediate filaments and microtubules are interconnected with the nucleus and surrounding organelles (Golgi apparatus, Mitochondria, rough and smooth endoplasmic reticulum). Nesprins (Ns) bind the nucleus with the actomyosin cytoskeleton. With the change in cell shape and contractility there is an alteration to spatial localization of organelles which may lead to a conformational change in the nuclear pores. (D) - Mechanotransduction at the focal adhesion (FA). (D, LEFT): Nascent adhesions (NA), focal complexes 8
Page 1 and 2: DEPARTAMENTO DE CIÊNCIAS DA VIDA F
Page 3 and 4: ii Queria agradecer à minha namora
Page 5 and 6: iv isolation of hUCM-MSCs was ever
Page 7 and 8: vi Conseguimos optimizar um novo pr
Page 9 and 10: viii Table of contents Acknowledgem
Page 11 and 12: x III. 6 - Influence on MSCs specif
Page 13 and 14: xii Figure 2| Proteins related to t
Page 15 and 16: xiv the change in distribution with
Page 17 and 18: xvi Cells were stained with anti-B-
Page 19 and 20: xviii List of abbreviations AA - Ac
Page 22: 1 Chapter I Introduction
Page 25 and 26: 4 There are many advantages in usin
Page 27: 6 only ones MSCs can differentiate
Page 31 and 32: 10 1C)(Dogterom et al., 2005). Not
Page 33 and 34: 12 extracellular ligand and the cyt
Page 35 and 36: 14 Figure 3 - The Rigidity Sensing
Page 37 and 38: 16 Figure 5 - Images depicted on A)
Page 39 and 40: 18 Figure 7 - A) Immunofluorescence
Page 41 and 42: 20 (GFAP) (is a class-III intermedi
Page 43 and 44: 22 can specify lineage towards cell
Page 45 and 46: 24 I.3 - Project rationale and expe
Page 47 and 48: 26 Figure 12 - MSCs cultured on 1 a
Page 49 and 50: 28 than that in soft substrate. Thi
Page 52: 31 Chapter II - Materials and Metho
Page 55 and 56: 34 Covance and DakoCytomation, resp
Page 57 and 58: 36 clone SAM1; mouse Pe-Cy7 anti-hu
Page 59 and 60: 38 To functionalize the complete su
Page 62: 41 Chapter III Results
Page 65 and 66: 44 2008) and also as previously est
Page 67 and 68: 12.5% Ac PBS Fold increase of hMSCs
Page 69 and 70: ≈12 kPa / COL-1 +FN ≈12 kPa / C
Page 71 and 72: 50 Figure 19 - Immunophenotype of U
Page 73 and 74: 52 coated with COL-1 (similar to wh
Page 75 and 76: 54 hydrogels of 1 and 7 kPa (for ce
Page 77: Figure 23 - hMSCs cultured on TCP (
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60 Discussion MSCs are widely used
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62 isolating MSCs on the PA hydroge
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64 2011) and expanded on TCPs for 5
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67 Conclusion In conclusion, we opt
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70 Cretu, A., Castagnino, P. & Asso
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72 McBeath, R. et al., 2004. Cell s
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74 Wang, N., Butler, J.P., and Ingb
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