The incidence rate of chronic hepatopathy in Taiwan is high, which afflicts the patients by progressively developing irreversible cirrhosis. Hepatic fibrosis is the intermediate and crucial stage of this process, characterized by reversibility. If treated properly in this stage, cirrhosis can be successfully prevented. In the liver, activated stellate cells are the key mediators of fibrosis. Transforming growth factor-

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Liver fibrosis, a major medical problem with significant morbidity and mortality, is considered as a wound-healing response to a variety of chronic stimuli. It is characterized by an excessive deposition of extracellular matrix (ECM) proteins, which disrupts the normal architecture of liver and ultimately leads to pathophysiological damage to liver. Hepatoma-derived growth factor (HDGF), a growth factor originally purified from hepatoma cells, is highly expressed in fetal hepatocytes and hepatoma. It is known to play multifunctional roles in mitogenesis, organogenesis, embryogenesis, and tumorigenesis. Its expression correlates with the proliferating state of hepatocellular carcinoma (HCC) and serves as a prognostic factor. Since liver fibrosis frequently occurs prior to HCC development, the specific aim of this study is to investigate the role of HDGF in the progression of liver fibrosis by using animal models of mice receiving either bile duct ligation surgery or carbon tetrachloride administration. Quantitative real-time PCR and Western blotting analysis showed a significant elevation of HDGF expression in both models. HDGF levels correlated with progression of liver fibrosis in a time-dependent manner as well as paralleled with the expression of other two fibrotic markers, transforming growth factor-b1 (TGF-b1) and pro-collagen type I, in fibrotic livers. Intriguingly, the over-expressed HDGF protein was localized mainly in perivenous hepatocytes of fibrotic livers. Besides, adenovirus-mediated HDGF gene delivery potentiated the production of TGF-b1 and pro-collagen type I, thereby enhancing the intrahepatic collagen matrix deposits as evidenced by Sirius red stain and morphometrical analysis. In cultured hepatocytes, TGF-b1 and HDGF mutually up-regulated their de novo synthesis only when grown on collagen-coated matrix, strongly suggesting that the TGF-b1- and/or HDGF-driven pro-fibrogenic signaling is collagen-dependent and a vicious circle may exist at the initial stage of hepatic fibrogenesis. Moreover, administration with recombinant HDGF stimulated BrdU uptake and synthesis of both a-smooth muscle actin and pro-collagen type I in cultured hepatic stellate cells, implicating that a mode of paracrinal action lies between these two cell types. In conclusion, HDGF plays a pro-fibrogenic role during liver fibrosis and blockade of HDGF pathway may potentially constitute the preventive or therapeutic strategies for chronic liver diseases.

Our aim was to understand mechanisms responsible for excessive electrolyte loss in the sweat gland and the potential impact on fluid balance during exercise in heat stress conditions. Human physiological testing under exercise/heat stress and immunofluorescence staining of sweat glands from skin biopsies were compared between healthy individuals (with normal and high sweat sodium chloride concentration, [NaCl]) and with cystic fibrosis patients (CF), who exhibit excessively salty sweat due to a defect of Cl- channel cystic fibrosis transmembrane conductance regulator (CFTR). Three novel findings are presented. First, excessively salty sweat may be associated with reduced expression of CFTR in the sweat gland reabsorptive duct of healthy individuals in addition to in those with CF; however, although a link to a CF gene mutation in healthy individuals with high sweat [NaCl] was not demonstrated, the possibility of an undetected CFTR mutation or polymorphism remains to be investigated as an underlying mechanism. Two, CF and healthy individuals with excessively salty sweat respond to moderate dehydration (3% body weight loss during exercise) with an attenuated rise in serum osmolality, greater relative loss in plasma volume, but similar perceived thirst compared to healthy individuals with "normal" sweat [NaCl]. However, individuals with CF respond to rehydration with hypotonic beverage by drinking less ad libitum in response to reduced serum [NaCl], suggesting that thirst-guided fluid replacement may be more appropriate for this population rather than restoring 100% of sweat loss following dehydration as is often recommended in healthy individuals.

Epithelial to mesenchymal transition (EMT), the transdifferentation of an epithelial cell into a mesenchymal fibroblast, is a cellular process necessary for embryonic development and wound healing. However, uncontrolled EMT can result in accumulation of myofibroblasts and excessive deposition of ECM, contributing to the pathological progression of fibrotic diseases such as pulmonary fibrosis. The ability to control EMT is important for development of novel therapeutics for fibrotic pathologies and for designing novel biomaterials for tissue engineering applications seeking to promote EMT for development of complex tissues. EMT is a highly orchestrated process involving the integration of biochemical signals from specific integrin-mediated interactions with extracellular matrix (ECM) proteins and soluble growth factors such as TGFβ. TGFβ, a potent inducer of EMT, is activated via cell contraction-mediated mechanical release of the growth factor from a macromolecular latency complex. Thus TGFβ activity and subsequent EMT may be influenced by the biochemical and biophysical state of the surrounding ECM. Based on these knowns, it was hypothesized that both changes in integrin engagement and increases in substrate rigidity would modulate EMT due to changes in epithelial cell contraction and TGFβ activation. Here we show that integrin-specific interactions with fibronectin (Fn) fragments displaying both the RGD and PHSRN binding sites facilitate cell binding through α5β1 and α3β1 integrins, and lead to maintenance of epithelial phenotype, while Fn fragments displaying only the RGD site facilitate cell binding through αv integrins and lead to EMT. An in depth investigation into α3β1 binding to Fn fragments indicates that binding is dependent on both the presence and orientation of the PHSRN site. Studies investigating the contribution of ECM stiffening on EMT responses show that increasingly rigid Fn substrates are sufficient to induce spontaneous EMT. Analysis of TGFβ-responsive genes implicate TGFβ-expression, activation or signaling as a mechanism for the observed EMT responses. Together these results suggest that the ECM micromechanical environment is a significant contributor to the onset of EMT responses and provide insights into the design of biomaterial-based microenvironments for the control of epithelial cell phenotype.

Se estudiaron 236 historias clínicas de pacientes atendidos en el Hospital Edgardo Rebagliati Martins, desde el año 2000 hasta julio del 2004.con fibrosis o cirrosis hepática por hepatitis viral B y C con el objetivo de evaluar el valor de diagnóstico de la ultrasonografía en comparación con el diagnóstico histopatológico. Se aplicó un sistema de puntaje ultrasonográfico a cada paciente de acuerdo a los signos US clasificándolos en una escala de 4-11. Se transcribieron los puntajes histopatológicos según el sistema METAVIR y se evaluó la sensibilidad, especificidad, valor predictivo positivo y valor predictivo negativo para la puntuación US, compuesta de superficie, parénquima, borde hepático y pared porta. A partir del punto óptimo se hallaron gráficos de curvas ROC lo que permitió tener una mejor visión de las pruebas de diagnostico. Se halló que una puntuación US 6 fue el mejor punto de corte para la predicción de Cirrosis y fibrosis grado III relacionados a HBV y HCV, con sensibilidad, la especificidad, el valor predictivo positivo y el valor predictivo negativo de 97.2%, 71.4%, 89.9%, 90.95 respectivamente. Las puntuaciones US están altamente y significativamente correlacionado 0.73(P<0.01) con la histopatología. La ultrasonografía en el diagnostico de la fibrosis y cirrosis por hepatitis B y C, muestra un mayor valor en la sensibilidad (85.39), la especificidad (85.71) y exactitud (85.59) en relación a la superficie del hígado. Los resultados demuestran que la ultrasonografía es un examen confiable para el diagnostico de la cirrosis y la fibrosis hepática tanto para la hepatitis viral B y C, aunque con mayor predicción para la hepatitis viral B. Los datos permiten concluir que la fibrosis y la cirrosis por hepatitis B y C, pueden ser correctamente diagnosticadas con un 85.59% de exactitud empleando un solo signo ultrasonográfico (superficie del hígado).

Studies of widely diverse species of a protein are a powerful tool to gain information on the structure and function of the protein. We investigated the response of human, pig, shark and killifish cystic fibrosis trans-membrane conductance regulator (CFTR) to specific inhibitors of the channel: CFTRinh-172, GlyH-101, and glibenclamide. In several expression systems, including isolated perfusions of the rectal gland, primary cell cultures of rectal gland tubules and oocyte expression, we observed fundamental differences in the sensitivity to inhibition by these CFTR blockers. We used primarily two-electrode voltage clamping of cRNA microinjected Xenopus laevis oocytes. In oocyte studies, shark CFTR was insensitive to CFTRinh-172 (maximum inhibition 8 ± 1.4% at 20µM), pCFTR was insensitive to Glibenclamide (maximum inhibition 12.8 ± 4.2% at 200µM), and all species were sensitive to GlyH-101 (maximum inhibition with pCFTR of 80.2 ± 3.6% at 20µM). Shark CFTR was completely insensitive to inhibition by CFTRinh-172 in short circuit current experiments (2.5 ± 0.15 % inhibition of chloride secretion) compared to inhibition with GlyH-101 (56.5 ± 6.56 % inhibition of chloride secretion). Perfusion studies confirmed these results. These experiments demonstrate a profound difference in the sensitivity of different CFTR species to inhibition by CFTR blockers. However, the amino acid residues that have been proposed by site directed mutagenesis studies to be responsible for inhibitor binding are uniformly conserved in all four isoforms studied. Therefore, the differences cannot be explained by simply targeting one amino acid for site-directed mutagenesis. Rather, the potency of the inhibitory actions of CFTRinh-172, Gly-H101 and glibenclamide on the CFTR molecule is dictated by the local environment and the three dimensional structure of residues that form the vestibule and the chloride pore.

Thesis (M.Sc.)--University of Alberta, 2010. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Master of Science in Clinical Epidemiology, Department of Public Health Sciences. Title from pdf file main screen (viewed on February 23, 2010). Includes bibliographical references.

Fibrosis of vital organs remains one of the leading causes of death in the developed world, where it occurs predominantly in soft tissues (liver, lung, kidney, heart) through fibroblast proliferation and deposition of extracellular matrix (ECM). In the process of fibrosis, remodeling and deposition of ECM results in stiffening of cellular microenvironment; cells also respond to these changes in the stiffness through engagement of their cytoskeleton and signaling via cell-ECM contacts. Thus, understanding to what extent the stiffness of the cellular microenvironment changes as a consequence of fibrotic progression, and how cells respond to this change, is critical. In this thesis, we quantitatively measured stiffness of the lung parenchyma and its changes during fibrosis. We find that the average stiffness increases by approximately 10-fold. We then investigated how changes in ECM rigidity affect the cytoskeletal phenotype of lung fibroblasts. We find a complex relation between expression of the glycoprotein Thy-1 (CD90) and ECM rigidity-dependent cytoskeletal phenotype (i.e. “mechanotransduction”). Finally, we investigate a mechanism for the regulation of rigidity sensing by Thy-1 and its involvement in intracellular signaling through cell-ECM contacts. Taken together, this work helps define in vivo parameters critical to the fibrogenesis program and to define unique cellular phenotypes that may respond or contribute to mechanical homeostasis in fibrotic diseases.

The Gram-negative bacterium Pseudomonas aeruginosa is a notorious opportunistic pathogen of individuals with the genetic disease cystic fibrosis (CF). Pseudomonas aeruginosa establishes a chronic infection within the CF lung, where the sputum accumulation characteristic of CF provides a complex and copious growth substrate. P. aeruginosa can grow to high densities in vivo (>10⁹ cells/ml lung sputum), and exacerbations associated with P. aeruginosa high density in vivo growth are primary contributors to CF morbidity and mortality. Surprisingly little is known about the catabolic processes that underlie P. aeruginosa in vivo growth. Unfortunately, nutritional modeling of the CF lung environment in animal models is difficult, as current animal models fail to mimic the sputum accumulation characteristic of CF. In this dissertation, I describe the use of expectorated CF sputum as a P. aeruginosa in vitro growth medium. Using global expression analysis, I show that P. aeruginosa up-regulates genes important for amino acid and lactate metabolism during growth in CF sputum as compared to a laboratory medium. P. aeruginosa also demonstrates enhanced production of the cell-cell communication signal 2-heptyl-3-hydroxy-4-quinolone (the Pseudomonas quinolone signal, PQS), a critical regulator of virulence factor production, during growth in CF sputum. Further, I use chemical analyses of CF sputum samples to develop a defined, synthetic medium that can be used to nutritionally model in vivo conditions. Using this medium, I show that PQS biosynthesis and aromatic amino acid metabolism are intimately linked and that cell-cell communication mediated by PQS is strikingly dependent upon the growth environment of P. aeruginosa. In addition, I demonstrate that P. aeruginosa preferentially consumes specific carbon sources present in the CF sputum milieu during rapid growth. I also describe the use of in vivo-relevant nutrient concentrations to evaluate the potential for P. aeruginosa anaerobic growth in CF sputum. Finally, I describe the purification and characterization of the aromatic amino acid-responsive transcriptional regulator PhhR and discuss its potential role in regulation of P. aeruginosa in vivo carbon substrate preference. / text