Liver fibrosis is a wound healing process in liver with¡@chronic injury and is characterized by the excess production and accumulation of extracellular matrix (ECM) component. Liver injury of any etiology may lead to activation of hepatic stellate cells (HSCs), which are trans-differentiated from lipocyte-like cells to highly proliferative myofibroblast-like cells. Activation of HSCs is considered a crucial event that promotes increased ECM production and consequently hepatic fibrosis. Liver fibros is resulted from a net increased synthesis and decreased degradation of ECM proteins. Pluchea indica (Less) has been reported to have antipyretic, anti-ulcer, anti-inflammatory, anti-oxidant, diuretic and anti-amoebic activities. Our previous studies showed that the aqueous extract of roots from P. indica (PIRAE) showed that it can suppress the growth and migration of HeLa and GBM8401 cancer cell lines, and also significantly reduce serum glutamate pyruvate transaminase (GPT), alpha-smooth muscle actin (£\-SMA) and collagen type I expression in animal model of liver fibrosis induced by thioacetamide (TAA). In this study, we plan to investigate the effects of PIRAE on activation, proliferation and migration of rat culture activated HSCs. The results indicated that protein expression of £\-SMA and collagen type I of HSCs was decreased followed by treatment of either 0.5 or 1.0 mg/ml PIRAE for 48 hours. In addition, the effects of PIRAE on proliferation in culture activated HSCs were assessed by analyses of cell growth curve, MTT, WST-1 and BrdU, respectively. The results showed that PIRAE inhibited HSCs proliferation in a dose- and time-dependent manner. Moreover, wound healing assay and transwell assay showed that PIRAE prevented migration in activated HSCs. In conclusion, PIRAE may suppresse culture activated HSCs proliferation, migration, and activation of culture activated HSCs, as well as accumulation of collagen type I.

Thesis (M.S.)--University of Hawaii at Manoa, 2003. / Includes bibliographical references (leaves 49-58). Also available via World Wide Web.

The objective of the presented research is to examine the relationship between the cellular microenvironment and biochemical response of metastatic cells. Clinically recognized as a trait of cancer progression, the cellular microenvironment can have variable and distinct mechanical properties that are processed via cellular mechanosensing, resulting in a cellular biochemical response. A range of studies investigating the interactions between the cellular micromechanical environment and the cell's molecular response during disease progression have been made, yet remain absent of quantitative characterization of many of these coordinated responses. The fundamental inquiry that drives the following research attempts to elucidate how a cell perceives the physical microenvironment and converts that signal to a biochemical response. With the goal of providing insight to such responses, the presented research seeks to elucidate the following questions: (1) What are the integrated effects of ECM stiffness, ECM architecture, and breast cancer cell metastatic potential on cell migration? (2) How does endogenous tissue transglutaminase (tTG) cross-linking of the ECM scaffold effect ECM mechanical properties? (3) How does the architecture and stiffness of the extracellular matrix (ECM) effect the systems-level cellular migration and signaling response? (4) What are the integrated effects of ECM architecture and the targeted knockdown of integrin [beta]1 and MT1-MMP on cellular metastatic potential? The presented research utilizes an interdisciplinary approach, integrating experimental mechanics, biochemical analysis, cellular biology techniques, covalent chemistry, and various microscopy techniques, to investigate these events. In short, cancerous cells are cultured atop or within synthetic collagen type I ECMs of varying mechanical stiffness and structure. These cells are subsequently analyzed by molecular analysis and immunoassays, including quantitative PCR, Western blotting, and gelatin zymography, to acquire measures of the cellular response to perturbations of micromechanical environment. Time-lapse microscopy experiments and subsequent image analyses enable observations of cellular migratory potential through synthetic ECMs. Results indicate that cooperative synergy between ECM properties, cell-matrix adhesion, and pericellular proteolysis drive cell migratory potential of highly invasive tumorigenic cell populations. Collectively, these findings contribute to the cancer biology and mechanobiology fields by systematically extending current insights of matrix mechanics, cellular signaling, and cellular migratory potential in cancer. / text

abstract / Biochemistry / Master / Master of Philosophy

The RET receptor tyrosine kinase is important during development of neural crest-derived tissues, particularly the enteric and sympathetic nervous systems, and in kidney morphogenesis. Activation of RET requires complex formation between a member of the Glial cell line-Derived Neurotrophic Factor family of ligands and a member of the GDNF-Family Receptor α co-receptors. Upon complex formation, RET becomes phosphorylated and subsequently activates multiple downstream signaling pathways, including those for cell-survival, differentiation, and migration. Mutations in RET can either interfere with or enhance normal RET signaling. Inhibiting RET mutations are associated with development of Hirschsprung disease, which is characterized by a lack of mature ganglia in the gut. Conversely, activating RET mutations are associated with several thyroid cancers. Papillary thyroid carcinoma is frequently associated with sporadic translocations between RET and other genes, known collectively as RET/PTC. A variety of heritable RET missense mutations lead to Multiple Endocrine Neoplasia type 2, which is associated with development of medullary thyroid carcinoma. Two cellular processes disrupted downstream of RET in these diseases are gene-expression and cell-migration. In order to clarify the effects of oncogenic mutations on gene-expression downstream of RET, we analyzed expression microarrays in a model using single mutant and isoform RET expression. We also examined the molecular mechanisms of cell-migration, using both functional cell-based assays and examination of integrins, cell-adhesion molecules important for cell-migration. Finally, we used a large cohort of thyroid tissues to examine RET and integrin expression. We showed that different forms of oncogenic RET do not affect transcription of different target genes, but rather target-gene transcription is proportional to phosphorylatability of mutant RET. We were also able to show that RET leads to activation of at least two integrin subunits (ITGB1 and ITGB3), and that they have unique activation patterns downstream of RET that correlate with cell-adhesion and migration. Finally, we showed that co-expression between RET, ITGB1, and ITGB3 is more frequent in malignant subtypes of thyroid tissues and that their co-expression is correlated to more aggressive thyroid cancer subtypes. Together, we have clarified how RET is able to mediate two important processes, gene-expression and cell-migration. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2011-11-08 13:53:32.474

Oligodendrocytes (OL) are the myelinating cells of the central nervous system (CNS). A series of complex cell signaling events in the CNS ensures successful myelination. Various molecular cues including growth factors, transcription factors and cytokines regulate myelination by inducing OL migration, proliferation and differentiation. Plateletderived growth factor A (PDGF-A) and fibroblast growth factor 2 (FGF2) are two of the most well characterized regulators of OP migration. The current study hypothesizes that PDGF-A and FGF2 regulate the migration of OP through transient activation of the extracellular signal-regulated protein kinase (ERK) signaling pathway. The results show that activation of ERK is required for OP migration. It also demonstrates the significance of threshold levels of growth factors and temporal regulation for OP migration. Furthermore, the chemokine CXCL1 has been shown to play a critical role in regulating the dispersal of OP during development, although the mechanisms underlying this regulation are unknown. Previous studies have shown that calcium flux is required for OP migration. CXCL1 induces calcium flux in cells; therefore we hypothesized that CXCL1 inhibition of OP migration was regulated via changes in intracellular calcium flux. However, our results show that CXCL1 inhibition of OP migration is independent of calcium signaling. In addition, we show that CXCL1 inhibition of OP migration is specific to PDGF-A induced migration. Lastly, the current study identifies a transcriptional regulator, methyl-CpG-binding protein 2 (MeCP2) as regulating the expression of myelin specific genes in a transgenic mouse. Interestingly, gene expression of myelin associated proteins myelin basic protein (MBP), myelin associated glycoprotein (MAG)and proteolipid protein (PLP), which play an important role in regulation of OL differentiation and subsequent formation of myelin of the myelin sheath, where found to be dysregulated. Overall, these findings reveal previously unknown roles of various intrinsic factors in successive phases of OL development. It aims to provide a better understanding of complexity to myelin development, function and disease.

Cells in vivo respond to chemical and mechanical cues in the environment. In fact, it is the resulting migration of cells as a cohesive group that underlies embryonic morphogenesis, wound repair and cancer tumour development and invasion. Techniques have been developed to investigate chemotaxis, haptotaxis and mechanotaxis – the directional movement of cells in response to soluble chemical cues, substrate-bound chemical cues and mechanical cues respectively. Most of the existing tools however, have been designed for and applied to the investigation of single cell migration. Given its importance in vivo, there is a need for adapting these methods and applying them to characterize directed collective cell migration. The main objective of my thesis was to engineer tools and quantitative methods to investigate collective cell migration and use them to compare single and collective migration in response to mechanical cues and substrate-adhered chemical cues in vitro.

Cells in vivo respond to chemical and mechanical cues in the environment. In fact, it is the resulting migration of cells as a cohesive group that underlies embryonic morphogenesis, wound repair and cancer tumour development and invasion. Techniques have been developed to investigate chemotaxis, haptotaxis and mechanotaxis – the directional movement of cells in response to soluble chemical cues, substrate-bound chemical cues and mechanical cues respectively. Most of the existing tools however, have been designed for and applied to the investigation of single cell migration. Given its importance in vivo, there is a need for adapting these methods and applying them to characterize directed collective cell migration. The main objective of my thesis was to engineer tools and quantitative methods to investigate collective cell migration and use them to compare single and collective migration in response to mechanical cues and substrate-adhered chemical cues in vitro.

Oligodendrocytes (OL) are the myelinating cells of the central nervous system (CNS). A series of complex cell signaling events in the CNS ensures successful myelination. Various molecular cues including growth factors, transcription factors and cytokines regulate myelination by inducing OL migration, proliferation and differentiation. Plateletderived growth factor A (PDGF-A) and fibroblast growth factor 2 (FGF2) are two of the most well characterized regulators of OP migration. The current study hypothesizes that PDGF-A and FGF2 regulate the migration of OP through transient activation of the extracellular signal-regulated protein kinase (ERK) signaling pathway. The results show that activation of ERK is required for OP migration. It also demonstrates the significance of threshold levels of growth factors and temporal regulation for OP migration. Furthermore, the chemokine CXCL1 has been shown to play a critical role in regulating the dispersal of OP during development, although the mechanisms underlying this regulation are unknown. Previous studies have shown that calcium flux is required for OP migration. CXCL1 induces calcium flux in cells; therefore we hypothesized that CXCL1 inhibition of OP migration was regulated via changes in intracellular calcium flux. However, our results show that CXCL1 inhibition of OP migration is independent of calcium signaling. In addition, we show that CXCL1 inhibition of OP migration is specific to PDGF-A induced migration. Lastly, the current study identifies a transcriptional regulator, methyl-CpG-binding protein 2 (MeCP2) as regulating the expression of myelin specific genes in a transgenic mouse. Interestingly, gene expression of myelin associated proteins myelin basic protein (MBP), myelin associated glycoprotein (MAG)and proteolipid protein (PLP), which play an important role in regulation of OL differentiation and subsequent formation of myelin of the myelin sheath, where found to be dysregulated. Overall, these findings reveal previously unknown roles of various intrinsic factors in successive phases of OL development. It aims to provide a better understanding of complexity to myelin development, function and disease.

No description available.