"A quantitative understanding of the complex interactions between cells, soluble factors, and the biological and mechanical properties of biomaterials is required to guide cell remodeling towards regeneration of healthy tissue rather than fibrocontractive tissue. The goal of this thesis was to elucidate the interactions between the boundary stiffness of three-dimensional (3D) matrix and soluble factors on valvular interstitial cell (VIC) phenotype with a quantitative approach. The first part of the work presented in this thesis was to characterize the combined effects of boundary stiffness and transforming growth factor-β1 (TGF-β1) on cell-generated forces and collagen accumulation. We first generated a quantitative map of cell-generated tension in response to these factors by culturing VICs within micro-scale fibrin gels between compliant posts (0.15-1.05 nN/nm) in chemically-defined media with TGF-β1 (0-5 ng/mL). The VICs generated 100 to 3000 nN/cell after one week of culture, and multiple regression modeling demonstrated, for the first time, quantitative interaction (synergy) between these factors in a 3D culture system. We then isolated passive and active components of tension within the micro-tissues and found that cells cultured with high levels of stiffness and TGF-β1 expressed myofibroblast markers and generated substantial residual tension in the matrix yet, surprisingly, were not able to generate additional tension in response to membrane depolarization signifying a state of continual maximal contraction. In contrast, negligible residual tension was stored in the low stiffness and TGF-β1 groups indicating a lower potential for shrinkage upon release. We then studied if ECM could be generated under the low tension environment and found that TGF-β1, but not EGF, increased de novo collagen accumulation in both low and high tension environments roughly equally. Combined, these findings suggest that isometric cell force, passive retraction, and collagen production can be tuned by independently altering boundary stiffness and TGF-β1 concentration. In the second part, by using the quantitative information obtained from the first part, we investigated the effects of dynamic changes in stiffness on cell phenotype in a 3D protein matrix, quantitatively. Our novel method utilizing magnetic force to constrain the motion of one of two flexible posts between which VIC-populated micro-tissues were cultured effectively doubled the boundary stiffness and resulted in a significant increase in cell-generated forces. When the magnetic force was removed, the effective boundary stiffness was halved and the tissue tension dropped to 65-87% of the peak value. Surprisingly, following release the cell-generated forces continued to increase for the next two days rather than reducing down to the homeostatic tension level of the control group with identical (but constant) boundary stiffness. The rapid release of tension with the return to baseline boundary stiffness did not result in a decrease in number of cells with α-SMA positive stress fibers or an increase in apoptosis. When samples were entirely released from the boundaries and cultured free floating (where tension is minimal but cannot be measured), the proportion of apoptotic cells in middle region of the micro-tissues increased more than five-fold to 31%. Together, these data indicate that modest temporary changes in boundary stiffness can have lasting effects on myofibroblast activation and persistence in 3D matrices, and that a large decrease in the ability of the cells to generate tension is required to trigger de-differentiation and apoptosis. "

Myostatin is a secreted growth factor of the transforming growth-factor $\beta$ (TGF$\beta$) superfamily, and a powerful negative regulator of muscle mass in vertebrates. As such, there is considerable interest in developing pharmacological agents which inhibit myostatin signalling in order to stimulate muscle growth in the context of pathological muscle wasting. Like other TGF$\beta$ family proteins, myostatin is biosynthesised as an inactive (latent) precursor protein which requires proteolytic processing to liberate the mature bioactive growth factor. To examine the molecular basis of pro-myostatin latency and the mechanism by which it is activated in the extracellular space, I have determined the crystal structure of unprocessed human pro-myostatin and studied the properties of the protein at various stages of activation. Crystallographic analysis of pro-myostatin reveals a unique domain-swapped dimeric structure, with an open V-shaped conformation distinct from the prototypical family member, TGF$\beta$1. Following cleavage of the prodomains by furin, pro-myostatin persists as a stable non-covalent complex which is resistant to the natural inhibitor follistatin and exhibits significantly weaker bioactivity than the mature growth factor. A number of distinct structural features combine to stabilise the interaction between pro and mature domains and in doing so confer latency to the pro-complex. This facilitates a controlled, step-wise process of activation in the extracellular space and contributes to a complex network of regulatory control. The results presented here provide a structural basis for understanding the effect of natural polymorphisms on myostatin function and a starting point for structure-guided development of next generation myostatin inhibitors. As a proof-of-concept, I present preliminary data on prodomain derived stapled peptides as inhibitors of myostatin signalling.

Cells adhere to each other and to the extracellular matrix (ECM) through protein molecules on the surface of the cells. The breaking and forming of adhesive bonds, a process critical in cancer invasion and metastasis, can be influenced by the mutation of cancer cells. Several molecules have been reported to play a crucial role in cellular adhesion and proliferation, and eventually in cancer progression, with TGF-β being one of the most important. In this thesis, we propose a general framework to model cancer cells movement and aggregation, in response to nonlocal social interactions (that is, attraction towards neighbours that are far away, repulsion from those that are near by, and alignment with neighbours at intermediate distances), as well as other molecules' effect, e.g., TGF-β. We develop nonlocal mathematical models describing cancer invasion and metastasis as a result of integrin-controlled cell-cell adhesion and cell-matrix adhesion, for two cancer cell populations with different levels of mutation. The models consist of nonlinear partial differential equations, describing the dynamics of cancer cells and TGF-β dynamics, coupled with nonlinear ordinary differential equations describing the ECM and integrins dynamics. We study our models analytically and numerically, and we demostrate a wide range of spatiotemporal patterns. We investigate the effect of mutation and TGF-β concentration on the speed on cancer spread, as well as the effect of nonlocal interactions on cancer cells' speed and turning behaviour.

Epithelial to Mesenchymal Transition (EMT) is a dynamic process by which a distinct change in the phenotype and function of epithelial cells render them as mesenchymal cells. Characteristics of mesenchymal cells include the ability to invade, increased migratory kinetics and heightened resistance to apoptosis. Therefore, there is a strong need to fully understand the mechanism for the induction of EMT in pathological conditions such as carcinoma progression. Recent advances highlight two pivotal contributors, soluble growth factor (gf) signals, and mechanical signals, in the process. However, to date, no clear mechanism exists linking the two in epithelial transdifferentiation. Transforming Growth Factor-β1 (TGF-β1), a gf known to induce EMT in breast cancer formation, induces EMT on rigid surfaces and apoptosis on compliant surfaces. It is our belief that a combination of mechanical signals, gf signals, and the type of extracellular matrix (ECM) proteins assembled by cells together drive the process of EMT. Here we investigated the role of the ECM protein fibronectin (FN) in EMT. Upon assembly into elastic, insoluble fibrils through cell-generated forces which become larger on stiffer surfaces, FN is able to serve as a gf delivery system. We examined the following hypothesis: Increased tissue stiffness drives FN assembly, which exposes cryptic binding sites for various gfs, such as TGF-β1, and creates a high concentration of these gfs at the cell surface, which in turn drives EMT. In this project we investigated three aims: (1) evaluate the effect of inhibiting FN fibrillogenesis and gf localization on TGF-β1-induced EMT, (2) assess the effect of TGF-β1 concentration on spatial patterning of ECM dynamics, cell phenotype and adherens junctional force, and (3) probe the role of the FN matrix in TGF-β1-induced spatial patterning of EMT. Results showed that both inhibition of FN fibril assembly and blocking the gf binding site on fibrils significantly attenuated the downstream effects of EMT. In microcontact patterns of epithelial colonies, increasing gf concentration led to spatial patterning of FN fibrils, cell phenotype and cell-cell junctional force. Elimination of FN fibrils effectively attenuated TGF-β1-induced spatial patterning. The knowledge acquired through these studies serves as an addition to an increasingly important body of work aimed at elucidating how physical changes within the microenvironment regulate physiology and pathology. By establishing a novel mechanism by which gf signaling induces EMT through interaction with the extracellular matrix, this research serves to combat the development and initiation of pathological phenomena, such as metastasis.

<p>Mast cells are essential effector cells in the immune system as they release several inflammatory mediators. An accumulation of mast cells has been described in inflammatory conditions such as asthma and allergic rhinitis. Increased mast cell number, in the skin and other organs, is also a characteristic in mastocytosis, a disease without an effective treatment. One explanation for the increase in mast cell number is migration of mast cells in the tissue. In our studies we utilised mast cell lines, including HMC-1; cell lines transfected with the <i>c-kit</i> gene; and <i>in vitro</i> developed mast cells.</p><p>Our aim was to characterise, two variants of the HMC-1 cell line; the signalling pathways essential for mast cell migration towards TGF-β and SCF; and the mechanism regulating mast cell accumulation in mastocytosis.</p><p>Our results help to explain inconsistent findings regarding mast cell biology when HMC-1 cells have been used as a model system. The two variants, which we name HMC-1⁵⁶⁰ and HMC-1^{560, 816}, are used in different laboratories around the world. HMC-1⁵⁶⁰ and HMC-1^{560, 816} exhibited different characteristics regarding their karyotype, phenotype as well as their set of activating point mutations in the Kit receptor. Furthermore, divergent signalling pathways are of importance for mast cell migration towards TGF-β and SCF. The classical MAP kinase-signalling cascade was found to be of major relevance for TGF-β-induced migration. In contrast, this pathway had a modest impact on SCF-induced migration, which instead was highly dependent on p38 MAP kinase signalling. Finally, one mechanism for mast cell accumulation in mastocytosis appeared to be an activating point mutation in the gene for the Kit receptor. This mutation appeared to prone transfected cells and mast cell progenitors to a higher rate of migration towards SCF if compared with cells expressing wt Kit receptor.</p><p>In conclusion, our results show the importance of two different MAP kinase signalling pathways and mutations in the Kit receptor for mast cell migration induced by various types of stimuli. This knowledge helps us to understand the mechanism </p>

<p>Anaplastic thyroid carcinoma (ATC) is an aggressive malignancy in man with stromal fibrosis as one of the main features. Carcinoma cells synthesized no or little collagen I protein. Pro-α1(I) collagen mRNA was expressed by stromal cells throughout the tumor, but expression of procollagen type I protein was restricted to stromal cells situated close to nests of carcinoma cells. These data suggest that the carcinoma cells stimulated collagen type I deposition by increasing pro-α1(1) collagen mRNA translation. </p><p>Cocultures, of the human ATC cell line KAT-4, with fibroblasts under conditions that allow the study of stimulatory factors on collagen mRNA translation, showed that the KAT-4 cells stimulated collagen type I protein synthesis in fibroblasts. Specific inhibitors of PDGF and TGF-β1 and -β3 were able to inhibit this carcinoma cell-induced stimulation of collagen type I synthesis. These findings suggest that tumor cells were able to stimulate collagen mRNA translation in stromal fibroblasts by, at least in part, transferring PDGF and/or TGF-β1 and -β3.</p><p>Xenograft transplantation of different ATC cell lines into athymic mice demonstrated that the low collagen producing carcinoma cell lines were less tumorigenic compared to non-collagen producing carcinoma cell lines. The morphology of tumors derived from non-collagen producing ATC cell lines showed a well demarked stroma surrounding carcinoma cell nests. </p><p>TGF-β1 and -β3 were found to play a role in generating a high tumor interstitial fluid pressure (TIPF) in experimental KAT-4 tumors. A specific inhibitor of TGF-β1 and -β3 was able to lower TIPF and reduce tumor growth after a prolonged period of treatment, suggesting that TGF-β1 and -β3 have a role in maintaining a stroma that support tumor growth.</p>

<p>TGF-β is the founding member of a multifunctional family of cytokines that regulate many aspects of cell physiology, including cell growth, differentiation, motility and death and play important roles in many developmental and pathological processes. TGF-β signals by binding to a heterotetrameric complex of type I and type II serine/threonine kinase receptors. The type I receptor is phosphorylated and activated by the type II receptor and propagates the signal to the nucleus by phosphorylating and activating receptor-regulated Smad proteins (R-Smads). Once activated, the R-Smads translocate to the nucleus together with the common partner Smad, Smad4, in heteromeric complexes and regulate transcription of target genes.</p><p>The cell cycle inhibitor p21^Waf1/Cip1 (p21) is induced by a number of factors including p53 and TGF-β, and its high expression is associated with cellular differentiation and senescence. Low levels of p21 are required for the propagation of the cell cycle, where high levels of p21 expression result to cell cycle arrest. The mode of action of p21 is by interacting with and dissociating cyclin E- and cyclin A-CDK complexes. p21 is very potently upregulated by TGF-β in cell types of epithelial origin and this sustained upregulation is of utmost importance for TGF-β to exert its growth inhibitory effect.</p><p>The aim of this study was to clarify the mechanisms by which the cell cycle inhibitor p21 is regulated during the TGF-β-induced cell growth inhibition. During the course of this work we established that TGF-β regulates p21 via the Smad pathway at the transcriptional level and that upregulation of the p21 levels cannot be achieved in the absence of proper Smad signaling. This regulation is achieved by Smad proteins interacting with the transcription factor Sp1 at the proximal <i>p21</i> promoter region. We also established that p21 is regulated by all the TGF-β superfamily pathways as we showed that all type I receptors of the superfamily are able to upregulate p21. Despite that, we demonstrated that p21 induction by other members of the superfamily, such as BMPs, is not sufficient for growth suppression. This is because BMPs regulate additional genes such as <i>Id2</i> that counteract the effect of p21 on cell growth. Furthermore, we examined the homeobox gene <i>Meox2</i>, which is regulated by TGF-β, and established that this factor is important for the sustained p21 regulation and the cell growth inhibitory program exerted by TGF-β. Simultaneously, we examined the cross-talk between Notch and TGF-β signaling pathways and established a synergy between Notch and TGF-β during epithelial cell growth inhibition. We showed that TGF-β-induced growth arrest requires intact Notch signaling. Abrogation of Notch signaling results in a blockage of sustained p21upregulation, required for the TGF-β-induced growth arrest to occur.</p><p>This work contributes substantially to the mechanism of both immediate-early and prolonged-late regulation of p21 by TGF-β-superfamily pathways, leading to cell growth inhibition of epithelial cells.</p>

<p>The Mad/Max/Myc transcription factor network takes part in the control of vital cellular functions such as growth, proliferation, differentiation and apoptosis. Dimerization with the protein Max is necessary for the Myc-family of oncoproteins and their antagonists, the Mad-family proteins, to regulate target genes and carry out their intended functions. Myc functions as a positive regulator of proliferation, antagonized by the growth inhibitory Mad-proteins that potentially functions as tumor supprerssors. Deregulated Myc expression is found in a variety of tumors and signals negatively regulating Myc expression and/or activity could therefore be of potential use in treating tumors with deregulated Myc.</p><p>Our aim was to therefore to investigate possible negative effects on Myc expression and activity by growth inhibitory cytokines and by the Myc antagonists, the Mad-family proteins.Two different cellular model systems of neuronal and hematopoietic origin have been utilized for these studies.</p><p>Our results show that Mad1 is upregulated during induced neuronal differentiation of SH-SY5Y cells. Further, the growth inhibitory cytokine interferon-g (IFN-g) was shown to cooperate with retinoic acid (RA) and the phorbol ester TPA in inducing growth arrest and differentiation in N-<i>myc</i> amplified neuroblastoma cell lines. In contrast to treatment with either agent alone, the combined treatment of TPA+IFN-g and RA+IFN-g led to upregulation of Mad1 and to downregulation of N-Myc, respectively, thus correlating with the enhanced growth inhibition and differentiation observed after combination treatment. Ectopic expression of an inducible Mad1 in monoblastic U-937 cells led to growth inhibition but did not lead to differentiation or enhancement of differentiation induced by RA, vitamin D3 or TPA. In v-Myc transformed U-937 cells Mad1 expression reestablished the TPA-induced G1 cell cycle arrest, but did not restore differentiation, blocked by v-Myc. The growth inhibitory cytokine TGF-b was found to induce Mad1 expression and Mad1:Max complex formation in v-Myc transformed U-937 cells correlating with reduced Myc activity and G1 arrest. </p><p>In conclusion, our results show that the Myc-antagonist Mad1 is upregulated by growth inhibitory cytokines and/or differentiation signals in neuronal and hematopoietic cells and that enforced Mad1 expression in hematopoietic cells results in growth inhibition and increased sensitivity to anti-proliferative cytokines. Mad1 and cytokine-induced signals therefore seem to cooperate in counteracting Myc activity.</p>

The Mad/Max/Myc transcription factor network takes part in the control of vital cellular functions such as growth, proliferation, differentiation and apoptosis. Dimerization with the protein Max is necessary for the Myc-family of oncoproteins and their antagonists, the Mad-family proteins, to regulate target genes and carry out their intended functions. Myc functions as a positive regulator of proliferation, antagonized by the growth inhibitory Mad-proteins that potentially functions as tumor supprerssors. Deregulated Myc expression is found in a variety of tumors and signals negatively regulating Myc expression and/or activity could therefore be of potential use in treating tumors with deregulated Myc. Our aim was to therefore to investigate possible negative effects on Myc expression and activity by growth inhibitory cytokines and by the Myc antagonists, the Mad-family proteins.Two different cellular model systems of neuronal and hematopoietic origin have been utilized for these studies. Our results show that Mad1 is upregulated during induced neuronal differentiation of SH-SY5Y cells. Further, the growth inhibitory cytokine interferon-g (IFN-g) was shown to cooperate with retinoic acid (RA) and the phorbol ester TPA in inducing growth arrest and differentiation in N-myc amplified neuroblastoma cell lines. In contrast to treatment with either agent alone, the combined treatment of TPA+IFN-g and RA+IFN-g led to upregulation of Mad1 and to downregulation of N-Myc, respectively, thus correlating with the enhanced growth inhibition and differentiation observed after combination treatment. Ectopic expression of an inducible Mad1 in monoblastic U-937 cells led to growth inhibition but did not lead to differentiation or enhancement of differentiation induced by RA, vitamin D3 or TPA. In v-Myc transformed U-937 cells Mad1 expression reestablished the TPA-induced G1 cell cycle arrest, but did not restore differentiation, blocked by v-Myc. The growth inhibitory cytokine TGF-b was found to induce Mad1 expression and Mad1:Max complex formation in v-Myc transformed U-937 cells correlating with reduced Myc activity and G1 arrest. In conclusion, our results show that the Myc-antagonist Mad1 is upregulated by growth inhibitory cytokines and/or differentiation signals in neuronal and hematopoietic cells and that enforced Mad1 expression in hematopoietic cells results in growth inhibition and increased sensitivity to anti-proliferative cytokines. Mad1 and cytokine-induced signals therefore seem to cooperate in counteracting Myc activity.

Mast cells are essential effector cells in the immune system as they release several inflammatory mediators. An accumulation of mast cells has been described in inflammatory conditions such as asthma and allergic rhinitis. Increased mast cell number, in the skin and other organs, is also a characteristic in mastocytosis, a disease without an effective treatment. One explanation for the increase in mast cell number is migration of mast cells in the tissue. In our studies we utilised mast cell lines, including HMC-1; cell lines transfected with the c-kit gene; and in vitro developed mast cells. Our aim was to characterise, two variants of the HMC-1 cell line; the signalling pathways essential for mast cell migration towards TGF-β and SCF; and the mechanism regulating mast cell accumulation in mastocytosis. Our results help to explain inconsistent findings regarding mast cell biology when HMC-1 cells have been used as a model system. The two variants, which we name HMC-1560 and HMC-1560, 816, are used in different laboratories around the world. HMC-1560 and HMC-1560, 816 exhibited different characteristics regarding their karyotype, phenotype as well as their set of activating point mutations in the Kit receptor. Furthermore, divergent signalling pathways are of importance for mast cell migration towards TGF-β and SCF. The classical MAP kinase-signalling cascade was found to be of major relevance for TGF-β-induced migration. In contrast, this pathway had a modest impact on SCF-induced migration, which instead was highly dependent on p38 MAP kinase signalling. Finally, one mechanism for mast cell accumulation in mastocytosis appeared to be an activating point mutation in the gene for the Kit receptor. This mutation appeared to prone transfected cells and mast cell progenitors to a higher rate of migration towards SCF if compared with cells expressing wt Kit receptor. In conclusion, our results show the importance of two different MAP kinase signalling pathways and mutations in the Kit receptor for mast cell migration induced by various types of stimuli. This knowledge helps us to understand the mechanism