Thesis (Ph. D.)--University of Virginia, 2008. / Includes bibliographical references. Also available in electronic form as viewed 2/16/2009.

Cardiac fibroblast to myofibroblast phenoconversion is a critical step during the development of cardiac fibrosis. Myofibroblasts chronically remodel extracellular matrix that results in myocardial stiffening, cardiac dysfunction and eventually heart failure. Previously we showed that Meox2, a homeobox transcription factor, can inhibit myofibroblast phenoconversion. Here we show that Zeb2, a repressor of Meox2, plays a crucial role during this phenoconversion process. Zeb2 overexpression significantly upregulates the expression of three key myofibroblast markers: α-SMA, SMemb and ED-A fibronectin in primary rat cardiac myofibroblast. We show that Zeb2 is highly expressed in myofibroblast nuclei whereas it is minimally expressed in fibroblast nuclei. Zeb2 overexpression in myofibroblasts results in a less migratory and more contractile mature myofibroblast phenotype. Moreover, Zeb2 overexpression represses Meox2 expression in endothelial cells. Thus, the current study enhances our understanding of the mechanism behind myofibroblast phenoconversion and provides a basis for developing Zeb2-based novel anti-fibrotic drug in the future. / February 2016

Chronic exposure to solar UVA plays a causative role in skin photoaging and photocarcinogenesis. Guided by exploratory difference-in-gel-electrophoresis (DIGE)-proteomics, we identified the thiol-dependent cysteine-proteases cathepsin B and cathepsin L as novel UVA-targets undergoing photo-oxidative inactivation upstream of autophagic-lysosomal dysfunction. In human skin fibroblasts, exposure to noncytotoxic doses of chronic UVA (9.9 J/cm ², twice a week, 3 weeks) caused pronounced photooxidative impairment of cathepsin B and L enzymatic activity suppressed by antioxidant intervention. Western blot analysis revealed extensive 4-hydroxy-2-trans-nonenal (4-HNE) modification of cathepsin B in UVA-exposed fibroblasts. Consistent with lysosomal impairment, accumulation of cellular autofluorescent material colocalizing with lysosomes was observed by confocal fluorescence microscopy, and extensive deposition of lipofuscin was detectable by transmission electron microscopy (TEM). Lysosomal expansion was further evidenced by increased immunodetection of lysosomal associated membrane protein-1 (Lamp-1) and Lysotracker-based flow cytometric analysis. While lysosomal membrane integrity remained intact, autophagic blockade was suggested by accumulation of cellular protein levels of LC3-II and p62 (sequestosome 1) in UVA-exposed fibroblasts. Furthermore, UVA-exposure modulated transcriptional levels of p62 (sequestosome 1, SQSTM1), α-synuclein (SNCA), and transglutaminase-2 (TGM2). Strikingly, pharmacological cathepsin B/L inhibition using CA074Me mimicked UVA-induced accumulation of lipofuscin and autophagic-lysosomal proteins (Lamp-1, LC3-II, and p62), as well as changes at the transcriptional levels. In order to determine if UVA-induced lysosomal impairment requires single or dual inactivation of cathepsin B and/or L, we used a genetic approach (siRNA) to selectively downregulate enzymatic activity of these target cathepsins. Monitoring protein levels of Lamp-1, LC3-II, and p62, we observed that only dual genetic antagonism (targeting both CTSB and CTSL expression) could mimic UVA-induced autophagic-lysosomal alterations, whereas single knockdown (targeting CTSB or CTSL only) did not reproduce the UVA-induced phenotype. Similarly, TEM revealed massive accumulation of lipofuscin-containing lysosomal vesicles in fibroblasts only after CTSB/CTSL-double knockdown. Taken together, our data indicate for the first time that UVA impairs lysosomal function causing autophagic-lysosomal alterations downstream of cathepsin B/L enzymatic inactivation. This work provides evidence for a heretofore unrecognized 'double-hit' mechanism of UVA skin photodamage where primary photo-oxidative insult occurs simultaneously with impaired clearance of damaged molecules and organelles downstream of dual inactivation of cathepsin B and L.

This study examined the cytotoxic effects of STERIPLEX™ HC (sBioMed, Orem, UT) and sodium hypochlorite (NaOCl) on human fibroblast cells in vitro. Fibroblasts exposed to various concentrations of NaOCl or STERIPLEX™ HC were visualized via light microscopy. Dilutions of either NaOCl or STERIPLEX™ HC that did not appear to disrupt the integrity of the cells were recorded for further analysis. Cells were then cultured and grown to confluence in five separate plates. A void was created down the middle of each plate. If the cells were viable, cellular confluence was seen. If nonviable, confluence of the cells did not occur. Both disinfectants showed absolute kill at all concentrations above 1%. The cells treated with 0.1% NaOCl were found to be nonviable. However, at 0.1% STERIPLEX™ HC, the cells were viable and able to replicate, filling the void and returning to confluence.

Atualmente é crescente o número de pacientes utilizando drogas que visam a alteração da remodelação óssea. Doenças como osteoporose e tumores ósseos têm possibilidade de tratamento com a utilização dos antirreabsortivos. Entretanto tais medicamentos apresentam, entre outros, um efeito colateral muito nocivo: a osteonecrose dos maxilares (ONM), que consiste em uma lesão rara, mas grave, da mandíbula ou maxila caracterizada por necrose óssea exposta. O denosumab é uma droga antirreabsortiva que possui um mecanismo de ação diferente do encontrado nos bisfosfonatos (BFs), medicação amplamente usada e anterior ao denosumab, entretanto já mostra efeitos colaterais similares aos BFs em relação à ONM e para ambos os medicamentos a fisiopatogenia da doença ainda não está esclarecida pela literatura Este trabalho teve como objetivo avaliar o efeito de diferentes concentrações do denosumab sobre a viabilidade, proliferação e migração de fibroblastos em cultura. Foram utilizados fibroblastos de mucosa bucal humana linhagem FMM1. Após serem submetidos aos testes de citotoxicidade com concentrações do denosumab variando de 10- 3?g a 10 - 7?g os fibroblastos não apresentaram quaisquer alterações quanto aos quesitos avaliados. Foi possível concluir que o denosumab não é citotóxico para fibroblastos em cultura. ecrose dos maxilares Fibroblastos / The number of patients using drugs that target the manipulation of bone remodeling is currently increasing. Bone volume diseases such as osteoporosis and tumors have the possibility of treatment with the use of antiresorptive medications. However, these drugs, among others, may present a very harmful side effect: osteonecrosis of the jaw (ONJ), which consists of a rare but severe injury characterized by exposed bone necrosis. The denosumab is an antiresorptive drug that presents a different mechanism of action found in bisphosphonates (BPs) and shows similar side effects to BPs regarding ONJ. BPs are a class of medication widely used and prior to denosumab. In both drugs the pathophysiology of the disease it is still not clear. This study aimed to evaluate the effect of denosumab in different concentrations on the viability, proliferation and migration of fibroblasts in culture. Were used human oral mucosa fibroblasts FMM1. After being subjected to denosumab concentrations ranging from 10-3?g to 10-7?g fibroblasts did not show any changes to the variables evaluated. It was possible to concluded that denosumab is not cytotoxic to fibroblasts in culture.

by Yeung Hiu Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 158-171). / Abstracts in English and Chinese. / ACKNOWLEDGEMENT --- p.i / ABBREVIATIONS --- p.ii / ABSTRACT (English & Chinese) --- p.iii / TABLE OF CONTENT --- p.viii / INDEX FOR FIGURES --- p.xii / INDEX FOR TABLES --- p.xvi / Chapter 1. --- INTRODUCTION --- p.2 / Chapter 1.1. --- History of Distraction Osteogenesis --- p.3 / Chapter 1.2. --- Clinical Application of Distraction Osteogenesis --- p.5 / Chapter 1.2.1. --- Limb-Lengthening --- p.5 / Chapter 1.2.2. --- Correction of Deformities and Non-Unions --- p.5 / Chapter 1.2.3. --- Bone Transport --- p.6 / Chapter 1.2.4. --- Reconstruction of the mandible --- p.7 / Chapter 1.3. --- Bone-specific Alkaline Phosphatase (BALP) --- p.8 / Chapter 1.4. --- Osteocalcin --- p.9 / Chapter 1.5. --- Bone Growth Factors --- p.11 / Chapter 1.6. --- Fibroblast Growth Factors (FGFs) --- p.12 / Chapter 1.6.1. --- Acidic Fibroblast Growth Factor (aFGF) --- p.13 / Chapter 1.6.2. --- Basic Fibroblast Growth Factor (bFGF) --- p.14 / Chapter 1.7. --- Transforming Growth Factor-pi (TGF-β1) --- p.16 / Chapter 1.8. --- Fracture Healing --- p.18 / Chapter 1.8.1. --- Histology --- p.18 / Chapter 1.8.2. --- Growth Factor Expression --- p.18 / Chapter 1.9. --- Distraction Osteogenesis --- p.19 / Chapter 1.9.1. --- Histology --- p.19 / Chapter 1.9.2. --- Growth Factor Expression --- p.20 / Chapter 1.10. --- Aim of the Study --- p.21 / Chapter 2. --- METHODOLOGY --- p.23 / Chapter 2.1. --- Animal Model --- p.23 / Chapter 2.1.1. --- Source of Animal --- p.23 / Chapter 2.1.2. --- Animal Operation --- p.23 / Chapter 2.1.3. --- Fracture Healing Model --- p.24 / Chapter 2.1.4. --- Distraction Osteogenesis Model --- p.24 / Chapter 2.2. --- Sample Collection --- p.25 / Chapter 2.2.1. --- Tissue Sample Collection and Preparation --- p.25 / Chapter 2.2.1.1. --- Test for the Complete Decalcification of the Calluses --- p.26 / Chapter 2.2.2. --- Blood Sample Collection and Storage --- p.26 / Chapter 2.3. --- Bone Mineral Density Measurement of the Distracted Callus and the Fracture Callus --- p.27 / Chapter 2.3.1. --- Fracture Healing Group --- p.27 / Chapter 2.3.2. --- Distraction Osteogenesis Group --- p.28 / Chapter 2.4. --- Serum Bone Specific Alkaline Phosphatase (BALP) Activity --- p.28 / Chapter 2.4.1. --- Wheat Germ Lectin (WGL) Precipitation of BALP --- p.28 / Chapter 2.4.1.1. --- Reagent --- p.28 / Chapter 2.4.1.2. --- Preparation and Measurement of Samples --- p.29 / Chapter 2.4.1.3. --- Auto-analyzer Setup --- p.30 / Chapter 2.5. --- Quantification of the Osteocalcin in Serum --- p.30 / Chapter 2.5.1. --- Reagent and Sample Preparation --- p.31 / Chapter 2.5.2. --- Detection Procedures --- p.31 / Chapter 2.6. --- Localization of the Growth Factors in Distraction Osteogenesis and Fracture Healing --- p.32 / Chapter 2.6.1. --- Immunohistochemistry of the Growth Factors --- p.33 / Chapter 2.6.1.1. --- Reagents and Solution Preparation --- p.33 / Chapter 2.6.1.2. --- Experimental Procedure --- p.36 / Chapter 2.6.1.3. --- Evaluation of Immunohistochmical Staining Results --- p.37 / Chapter 2.6.2. --- Verification of the Primary Antibody Used in the Study --- p.37 / Chapter 2.6.2.1. --- Tissue Preparation --- p.37 / Chapter 2.6.2.2. --- Antibody to Acidic Fibroblast Growth Factor (aFGF) --- p.38 / Chapter 2.6.2.2.1. --- Immunohistochemistry of Goat Brain and Growth Plate --- p.38 / Chapter 2.6.2.2.2. --- Dot Blot --- p.38 / Chapter 2.6.2.2.2.1. --- Materials and Reagents --- p.38 / Chapter 2.6.2.2.2.2. --- Procedures --- p.39 / Chapter 2.6.2.2.3. --- Sodium Dodecylsulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.41 / Chapter 2.6.2.2.3.1. --- Materials and Reagents --- p.41 / Chapter 2.6.2.2.3.2. --- Procedures --- p.42 / Chapter 2.6.2.2.4. --- Western Blotting --- p.43 / Chapter 2.6.2.2.4.1. --- Materials and Reagents --- p.43 / Chapter 2.6.2.2.4.2. --- Procedures --- p.44 / Chapter 2.6.2.3. --- Antibody to Basic Fibroblast Growth Factor --- p.45 / Chapter 2.6.2.4. --- Antibody to Transforming Growth Factor-β1 --- p.45 / Chapter 3. --- RESULTS --- p.53 / Chapter 3.1. --- Animal Model --- p.53 / Chapter 3.1.1. --- Fracture Healing Animal Model --- p.53 / Chapter 3.1.1.1. --- Radiography of Fracture Healing --- p.53 / Chapter 3.1.2. --- Distraction Osteogenesis Animal Model --- p.54 / Chapter 3.1.2.1. --- Gross Morphology of Distraction Osteogenesis --- p.54 / Chapter 3.1.2.2. --- Radiography of Distraction Osteogenesis --- p.55 / Chapter 3.2. --- Bone Mineral Density (BMD) Measurement --- p.56 / Chapter 3.2.1. --- In Fracture Healing --- p.56 / Chapter 3.2.2. --- Distraction Osteogenesis --- p.57 / Chapter 3.3. --- Bone-specific Alkaline Phosphatase Activity in Goat Serum --- p.59 / Chapter 3.3.1 --- ", Fracture Healing" --- p.59 / Chapter 3.3.2. --- Distraction Osteogenesis --- p.59 / Chapter 3.4. --- Serum Osteocalcin Measurement --- p.60 / Chapter 3.4.1. --- Fracture Healing --- p.60 / Chapter 3.4.2. --- Distraction Osteogenesis --- p.60 / Chapter 3.5. --- Histology --- p.61 / Chapter 3.5.1. --- Fracture Healing --- p.61 / Chapter 3.5.2. --- Distraction Osteogenesis --- p.64 / Chapter 3.6. --- Verification of Primary Antibody Used in the Study --- p.67 / Chapter 3.6.1. --- Antibody to Acidic Fibroblast Growth Factor --- p.67 / Chapter 3.6.1.1. --- Dot Blot --- p.67 / Chapter 3.6.1.2. --- Western Blotting --- p.68 / Chapter 3.6.1.3. --- Immunohistochemistry of Goat Brain and Growth Plate --- p.68 / Chapter 3.6.2. --- Antibody to Basic Fibroblast Growth Factor --- p.69 / Chapter 3.6.2.1. --- Dot Blot --- p.69 / Chapter 3.6.2.2. --- Immunohistochemistry of Goat Brain and Growth Plate --- p.69 / Chapter 3.6.3. --- Antibody to Transforming Growth Factor-β1 --- p.70 / Chapter 3.6.3.1. --- Western Blotting --- p.70 / Chapter 3.6.3.2. --- Immunohistochemistry of Growth Plate --- p.70 / Chapter 3.7. --- Localization of Growth Factors in Fracture Healing and Distraction Osteogenesis --- p.70 / Chapter 3.7.1. --- Acidic Fibroblast Growth Factor --- p.71 / Chapter 3.7.1.1. --- Fracture Healing --- p.71 / Chapter 3.7.1.2. --- Distraction Osteogenesis --- p.72 / Chapter 3.7.2. --- Basic Fibroblast Growth Factor --- p.73 / Chapter 3.7.2.1. --- Fracture Healing --- p.73 / Chapter 3.7.2.2. --- Distraction Osteogenesis --- p.74 / Chapter 3.7.3. --- Transforming Growth Factor-β1 --- p.75 / Chapter 3.7.3.1. --- Fracture Healing --- p.75 / Chapter 3.7.3.2. --- Distraction Osteogenesis --- p.76 / Chapter 4. --- DISCUSSION --- p.142 / Chapter 4.1. --- The Biochemical Events in Fracture Healing --- p.142 / Chapter 4.2. --- The Biochemical Events in Distraction Osteogenesis --- p.147 / Chapter 4.3. --- Limitations of the present study --- p.153 / Chapter 4.4. --- Future Study --- p.154 / Chapter 5. --- CONCLUSION --- p.156 / BIBLIOGRAPHY --- p.158

Mutations in FGFR2 are common in a subset of endometrial carcinomas. Given the emergence of small molecule inhibitors specific to this receptor tyrosine kinase, FGFR2 is an attractive therapeutic target. However, compensatory and adaptation mechanisms limit the clinical utility of compounds that target nodes in the receptor tyrosine kinase network. Here, we analysed the impact of FGFR inhibition in endometrial cancer cells and observed the emergence of a resistant population in an FGFR2-mutant cell line. To understand the mechanisms underlying this adaptation response, we used a phosphoproteomics approach to measure the kinase network in an unbiased manner. These experiments led to the identification of an AKT-related compensatory mechanism underpinning this resistance. Further dissection of this resistance mechanism utilising gene expression analysis showed PHLDA1, a negative regulator of AKT, was significantly down-regulated in resistant cells. This was further confirmed at the protein level. siRNA knockdown of PHLDA1 conferred immediate drug resistance in the FGFR2-mutant endometrial cancer cell line. Therefore, we identified PHLDA1 down-regulation as a mediator of drug resistance in FGFR2 mutant cancer cells, the first demonstration of the role of PHLDA1 in the acquisition and maintenance of drug resistance. Using a 3D physiomimetic model, we demonstrated that AKT inhibition alone also led to generation of a drug-resistant population. Most importantly, dual-drug therapy inhibited proliferation and induced cell death. Our data highlight how mass spectrometry and microarray gene expression analysis can complement each other in the identification of novel resistance mechanisms in cancer cells. These data suggest that combination treatment of FGFR2-mutant endometrial cancers, targeting both FGFR2 and AKT, represents a promising therapeutic approach.

A gene dosage study was conducted on a rare complete trisomy 22 human fibroblast cell line utilizing three lysosomal enzymes, ∝-iduronidase, ∝-galactosidase B, and arylsulfatase A, whose genes are located on chromosome 22 and two control enzymes, ,β-hexosaminidase A and -- fucosidase, with genes not on chromosome 22. A gene dosage effect was clearly demonstrated for an early passage number of the fibroblasts; however, later passage numbers gave inconclusive results. This study suggests that gene dosage studies must be carefully designed to be conducted only on early, matched passage number cells. ∝-fucosidase gave anomalous results most likely due to pleiotropic effects. The present gene dosage study confirmed the trisomic nature of the cell line studied and suggests that this type of study may be a useful diagnostic tool for small deletions, additions, or unbalanced translocations.

Thisbe (Ths) and Pyramus (Pyr) are the ligands for the Fibroblast-Growth-Factor (FGF)receptor Heartless (Htl), which is expressed in all mesodermal cells during gastrulation. To understand how these two FGFs orchestrate mesoderm spreading in gastrulation and mesoderm differentiation during organogenesis, loss and gain of function studies were performed. In an approach of functional analysis, a single mutant allele of ths was generated, ths759, for comparison of the single mutant conditions of ths and the null mesodermal cells to migrate and differentiate in a precise pattern.

The fetal lung undergoes extensive physiological and biochemical maturation prior to birth in preparation for its postnatal function as an organ for gas exchange. Pulmonary surfactant, a substance that reduces surface tension and prevents alveolar collapse, is produced by type II pneumocytes within the lung. Reduced ability to produce surfactant leads to neonatal respiratory distress syndrome. Synthesis of the phospholipid component of surfactant, phosphatidylcholine (PC), is stimulated by fibroblast-pneumocyte factor (FPF), a protein expressed by fibroblast cells within the fetal lung. Although its function is well known, the identity of this important protein has remained a mystery. Recent research has suggested that FPF may be neuregulin-1, a growth factor found in many tissues during development. Enhanced synthesis of PC (and therefore detection of FPF) is measured using a tissue culture-based method. Primary cultures of lung fibroblasts and type II pneumocytes are prepared, and fibroblast-conditioned medium (FCM) is exposed to the type II cells. Resultant PC synthesis is measured using radioisotope-labeled PC-precursor and a chloroform-based lipid extraction method. Initial results using this method were very inconsistent, so a study was undertaken to determine which parts of the method could be contributing to this inconsistency. Cell density of type II cultures (measured in μg DNA.plate-1) was shown to have a significant effect on results. Treatment of fibroblasts with 100 nM dexamethasone and exposure of type II cultures to the resultant FCM caused a mean 9.17% increase in PC synthesis, but when only type II cultures with a cell density below 25 μg DNA.plate-1 were analyzed, this value increased to 17.56%. Type II cultures with cell density above this threshold value showed a mean increase in synthesis of only 3.39%. The consistent application of [3H]-choline chloride also had a significant effect on results. Experiments utilizing phorbol 12-myristate 13-acetate to stimulate fibroblasts were very inconsistent. The mean activity of the initial [3H]-choline chloride solution prepared for these experiments was found to be 2.04 μCi.mL-1, compared to a mean of 4.79 μCi.mL-1 for all other experiments. Observations from this section of the study led to considerable revision of the method used to measure PC synthesis. Quadrupolar ion trap mass spectrometry (MS) was used to analyze FCM and determine if neuregulin-1 (NRG1) could be FPF. A mass spectrum was obtained for recombinant NRG1, with predominant ions of 1068, 1142 and 1246 m/z. All three of these ions were also detected in both control and dexamethasone-treated FCM. Partial fragmentation of 1068 m/z of NRG1 was achieved using MS2, and generated a base peak of 1047 m/z. This fragmentation was also observed in 1068 m/z from FCM. LC/MS was utilized to quantify NRG1 in FCM, using a standard curve generated using recombinant NRG1. Control FCM had a NRG1 concentration of 19.85 μg.mL-1, while the concentration in dexamethasone treated FCM was 41.59 μg.mL-1. FCM which had given no positive response to dexamethasone when tested using the indirect cultured cell system had a control NRG1 concentration of 20.85 μg.mL-1, and a dexamethasone treated concentration of 22.84 μg.mL-1. These values were not significantly different from the control value for FCM in those fibroblast cultures that had generated a positive response to dexamethasone. Results of this section of the study have provided strong evidence that NRG1 is a major component of FPF, and a review of the NRG1 signaling pathway further supports this conclusion. Insulin-like growth factors (IGFs) are functionally related to neuregulins and are known to be important in fetal development. The effect of IGF-II on synthesis of surfactant PC and its subsequent secretion from type II pneumocytes was studied. In terms of PC synthesis, IGF-II was tested at concentrations of 0.4, 0.6 and 0.8 μM. The mean increase in synthesis was found to be 6.00, 6.15 and 6.91%, respectively. These values were not significantly different from control values. Secretion of PC was tested over the concentration range of 0.1 to 1.6 μM, with no significant effect observed. Possible inhibition by IGF-II was also studied, using the known stimulants of secretion, neuromedin C and isoproterenol. No significant effect on the enhanced level of secretion was observed when IGF-II was added with either secretagogue. Lack of an appropriate receptor and/or the possibility that cultured cells may not exactly mimic the situation in vivo are probably the reasons IGF-II has no effect on either synthesis or secretion.