<p>The majority (75%) of renal cell carcinoma (RCC) is comprised of clear cell renal cell carcinoma (ccRCC). Despite ccRCC being the most aggressive form of RCC, our knowledge regarding the pathogenesis of the disease remains limited. We have identified upregulation of the extracellular protein periostin (POSTN) in ccRCC. Western blot analysis of ccRCC tumours from 27 patients demonstrated high POSTN protein expression in 26 tumours compared to their respective adjacent normal kidney tissue (ANK). Immunohistochemistry (IHC) analysis revealed high levels of stromal POSTN protein in the ccRCC primary tumours and 16 metastasized ccRCCs. Intriguingly, abundant stromal POSTN in the tumour and non tumour boundary were observed in local and metastaized ccRCC, and in A498 ccRCC cell-derived xenograft tumours. Collectively, these results suggest that the ccRCC-associated POSTN was derived from the stroma. This notion was supported by the co-existence of POSTN with α-smooth muscle actin (αSMA) in both local and metastasized ccRCC tumours. αSMA is a marker of activated stromal fibroblasts (myofibroblasts). Furthermore, co-culture of NIH3T3 murine fibroblasts with human A498 or 786-0 ccRCC cells dramatically enhanced POSTN transcription and secretion from NIH3T3 cells. Extracellular POSTN significantly enhanced A498 cell attachment. Upregulation of POSTN in NIH3T3 cells enhanced their proliferation. Taken together, my research demonstrates that 1) ccRCC induces fibroblast-mediated accumulation of extracellular POSTN, 2) stromal POSTN enhances ccRCC attachment, and 3) high levels of POSTN promotes fibroblasts' proliferation. These observations suggest a critical role for POSTN in mediating the co-evolving process between ccRCC and its stroma during ccRCC pathogenesis.</p> / Master of Science (MSc)

In order to examine the potential of HSV-1 as a vector to study the expression of DNA repair genes in mammalian cells, a recombinant virus containing the den V gene from bacteriophage T4 has been constructed. This gene encodes a pyrimidine dimer-specific endonuclease that has the capacity to initiate excision repair of DNA. Transfection studies indicate that excision repair deficient xeroderma pigmentosum (XP) group A cells are able to carry out excision repair initiated by the den V gene product. This gene along with the 3' LTR of Rous Sarcoma Virus and the SV40 polyadenylation signals were inserted into the non-essential glycoprotein I gene of HSV-1. Immunoprecipitation studies confirmed the production of the den V protein in virus infected cells. The uv survival of this HSV-1:den V recombinant virus was examined in various primary cell types. The cells examined in this study were primary fibroblasts from a normal individual, a Trichothiodystrophy patient and five XP patients as well as a mouse L cell line. The ability of the virally encoded den V gene to restore the excision repair deficiency in these cells was measured by monitoring the uv survival of HSV-1:den V as compared to wildtype HSV-1. Increased survival of HSV-1:den V was detected in Trichothiodystrophy cells, and in cells from XP complementation groups A, C and D, but not in XP cells from complementation groups E and F or in mouse L cells. These results demonstrate that HSV can be effectively used to study the expression of a cloned DNA repair gene in a variety of cell types. HSV has a substantial capacity of gene insertion and a wide host range including cells of human and rodent origin. / Thesis / Master of Science (MS)

Cancer-associated fibroblasts (CAFs) in the tumor microenvironment have been associated with formation of a dynamic and optimized niche for tumor cells to grow and evade cell death induced by therapeutic agents. We recently reported that ablation of β-catenin expression in stromal fibroblasts and CAFs disrupted their biological activities in in vitro studies and in an in vivo B16F10 mouse melanoma model. Here, we show that the development of a BRAF-activated PTEN-deficient mouse melanoma was significantly suppressed in vivo after blocking β-catenin signaling in CAFs. Further analysis revealed that expression of phospho-Erk1/2 and phospho-Akt was greatly reduced, effectively abrogating the activating effects and abnormal cell cycle progression induced by Braf and Pten mutations. In addition, the epithelial-mesenchymal transition (EMT)-like process was also suppressed in melanoma cells. Taken together, our data highlight an important crosstalk between CAFs and the RAF-MEK-ERK signaling cascade in BRAF-activated melanoma and may offer a new approach to abrogate host-dependent drug resistance in targeted therapy.

Introduction Cutaneous scars represent a common surgical complication, yet no effective drug therapy for scar treatment currently exists despite huge patient and physician demand. A connexin 43 (Cx43) carboxyl terminus (CT) mimetic peptide, alpha Connexin Carboxy-Terminus 1 (αCT1), has demonstrated efficacy in improving long-term scar appearance in pre-clinical and clinical trials. However, current understanding of the mechanism-of-action by which αCT1 improves long-term scar appearance with early intervention treatment is not well understood. Methods In vivo: Scar biopsies from 1) human, 2) Sprague-Dawley rat, and 3) IAF Hairless guinea pig trials of αCT1 were examined for collagen matrix structure at 4 weeks (all models), and 2 and 6 weeks (rat and guinea pig models only). Collagen matrix variables examined included local disorganization of the fibers, a variable that is higher in unwounded skin compared to scar tissue, and density of the fibers, which is higher in scar tissue but can also be used as an early temporal marker of the rate of healing. In vitro: Primary murine dermal fibroblasts were isolated from the whole dermis of 3-4 week old transgenic mice expressing collagen 1(α2) GFP-tpz. Cells were sorted for expression via FACS and plated on prealigned collagen substrate for 7 days under conditions favorable to generating extracellular matrix. Results: All in vivo scar biopsies demonstrated some level of altered collagen matrix structure with αCT1 treatment. Treated scars had higher local disorganization of the collagen fibers within the wound, and an increase in collagen matrix density compared to control at certain earlier timepoints that tended to decrease or disappear at later timepoints. The IAF Hairless guinea pig, a novel splinted wound healing model presented herein, was found to closely replicate the human dermal collagen profile and changes in collagen profile spurred by αCT1, significantly outperforming the traditional rat model. Primary dermal murine fibroblasts treated in vitro with αCT1 significantly increased synthesis of procollagen 1, the precursor of collagen 1 necessary for constructing the extracellular matrix, suggesting that at least part of the reason for higher collagen density at early in vivo timepoints is due to increased collagen synthesis by fibroblasts. Conclusion: αCT1 treatment in the early stages of wound healing prompts individual fibroblasts to increase their output of collagen and create a more disorganized early collagen matrix. These early changes potentially spur the long-term scar appearance improvements seen in clinical trials, and provide a basis for future work to discover the cellular pathways to alter in order to improve wound healing and cutaneous scarring outcomes. / Doctor of Philosophy / Skin wounds frequently result in scars that can range from barely visible to enormous eyesores. Almost everyone will experience at least one skin wound in their lifetime leading to a scar that they wish were less visible, feeding the multi-billion dollar market for anti-scarring agents. However, many of the products on store shelves that claim to reduce scar appearance have not proven those claims. Most of the therapies that do have some degree of scientific evidence to support their claims are difficult to use properly, such as silicone sheeting, and often result in only minor improvements to scar appearance. Alpha Connexin Carboxy-Terminus 1 (αCT1), marketed in clinical trials as Granexin® gel, is a protein-based therapy that works on the cellular level to fundamentally alter the skin's initial reaction to wounding and improving long-term scar appearance. This dissertation explores the link between cellular processes altered by αCT1 and long-term clinical improvements in scar appearance by studying both the extracellular matrix present in the scar in human and animal models and the creation of that extracellular matrix by dermal fibroblasts. In both human and animal models, topical application of αCT1 had no effect on skin surface appearance at early timepoints of 2-6 weeks, correlating with previous research that found scar appearance only improved at 3+ months post-injury. However, deep within the newly constructed tissue of the scar, these studies show the collagen organizational structure of αCT1-treated scars is more similar to unwounded skin and slightly more dense at early timepoints, suggesting αCT1 marginally improved the speed of healing. These findings in humans and animals were also verified in part in cell culture experiments that found dermal fibroblasts increased collagen output in response to αCT1 treatment. A novel wound healing model in the hairless guinea pig, superior at replicating human skin than established models like the rat, is also presented and shown to have effects strongly similar to the human with αCT1 treatment. These results provide a fundamental insight into the mode-of-action by which αCT1 may improve long term scar appearance and identifies early collagen structure as a target for future therapeutics to modify, as well as a new animal model in which to test them.

The objectives of this study were to: 1) develop a technique to analyze the <i>in vitro</i> cytotoxic activity of lymphocytes from adult horses against equine herpes virus-1 (EHV-1) infected allogenic equine dermal fibroblasts (EDF); 2) evaluate the ability of a 72 hour in vitro incubation with interleukin-2 (I L-2) to enhance the lymphocytic cytolytic activity against EHV-1 infected EDF; 3) compare the cytotoxic activity among lymphocytes isolated from pregnant mares and non-pregnant mares against EHV-1 infected EDF; 4) ascertain if any correlations existed between the percent cytotoxicity and percentage of lymphocytes phenotypically identified by five different mouse-anti-equine monoclonal antibodies; and 5) determine if any correlation existed between virus-neutralizing antibody titers and the percent cytotoxicity. Results of the study indicate that <i>in vitro</i> cytotoxic activity of equine lymphocytes against EHV-1 infected allogenic fibroblasts can be measured with a standard 4 hour 51Cr release assay. This activity was enhanced by an <i>in vitro</i> incubation with IL-2. The cytolytic activity of freshly isolated lymphocytes was greater for non-pregnant than pregnant mares. However, after IL-2 stimulation the cytolytic activity was greater for lymphocytes from pregnant mares. A positive correlation was not detected between the percentage of phenotypically identified cells and the percent cytoxicity, although several negative correlations were present. This suggests that the cytotoxic activity was either not mediated by any of the phenotypically identified cell populations or that the activity was mediated by several different cell populations. No correlation was detected between virus neutralizing antibody titers and the percent cytotoxicity. / Master of Science

Yes / Introduction Type 2 diabetes mellitus (T2DM) promotes adverse myocardial remodeling and increased risk of heart failure; effects that can occur independently of hypertension or coronary artery disease. As cardiac fibroblasts (CFs) are key effectors of myocardial remodeling, we investigated whether inherent phenotypic differences exist in CF derived from T2DM donors compared with cells from nondiabetic (ND) donors. Methods Cell morphology (cell area), proliferation (cell counting over 7-day period), insulin signaling [phospho-Akt and phospho-extracellular signal-regulated kinase (ERK) Western blotting], and mRNA expression of key remodeling genes [real-time reverse transcription-polymerase chain reaction (RT-PCR)] were compared in CF cultured from atrial tissue from 14 ND and 12 T2DM donors undergoing elective coronary artery bypass surgery. Results The major finding was that Type I collagen (COL1A1) mRNA levels were significantly elevated by twofold in cells derived from T2DM donors compared with those from ND donors; changes reflected at the protein level. T2DM cells had similar proliferation rates but a greater variation in cell size and a trend towards increased cell area compared with ND cells. Insulin-induced Akt and ERK phosphorylation were similar in the two cohorts of cells. Conclusion CF from T2DM individuals possess an inherent profibrotic phenotype that may help to explain the augmented cardiac fibrosis observed in diabetic patients.

No / Werner's syndrome (WS) is an autosomal recessive genetic disorder caused by loss of function mutation in wrn and is a useful model of premature in vivo ageing. Cellular senescence is a plausible causal mechanism of mammalian ageing and, at the cellular level, WS fibroblasts show premature senescence resulting from a combination of telomeric attrition and replication fork stalling. Over 90% of WS fibroblast cultures achieve < 20 population doublings (PD) in vitro compared to wild type human fibroblast cultures. It has been proposed that some cell types, capable of proliferation, will fail to show a premature senescence phenotype in response to wrn mutations. To test this hypothesis, human dermal keratinocytes (derived from both WS and wild type patients) were cultured long term. WS Keratinocytes showed a replicative lifespan in excess of 100 population doublings but maintained functional growth arrest mechanisms based on p16 and p53. The karyotype of the cells was superficially normal and the cultures retained markers characteristic of keratinocyte holoclones (stem cells) including p63 expression and telomerase activity. Accordingly we conclude that, in contrast to WS fibroblasts, WS keratinocytes do not demonstrate slow growth rates or features of premature senescence. These findings suggest that the epidermis is among the tissue types that do not display symptoms of premature ageing caused by loss of function of wrn. This is in support that Werner's syndrome is a segmental progeroid syndrome.

Tse, Chi Lok. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 116-131). / Abstracts in English and Chinese. / Declaration --- p.i / Abstract --- p.iii / Abstract in Chinese --- p.v / Acknowledgements --- p.vi / Table of Contents --- p.vii / List of Figures --- p.X / List of Tables --- p.xii / List of Abbreviations --- p.xiii / Chapter CHAPTER 1 --- General Introduction / Chapter 1.1 --- Regenerative Medicine --- p.1 / Chapter 1.2 --- Embryonic Stem Cells and Reprogramming --- p.3 / Chapter 1.3 --- Transdifferentiation --- p.6 / Chapter 1.4 --- The Cerebellum --- p.7 / Chapter 1.4.1 --- Functions of the cerebellum --- p.7 / Chapter 1.4.2 --- Structure and organization of the cerebellum --- p.8 / Chapter 1.4.3 --- Principle cellular components in the cerebellum --- p.12 / Chapter 1.4.3.1 --- Purkinje cells --- p.12 / Chapter 1.4.3.2 --- Granule cells --- p.12 / Chapter 1.4.3.3 --- Mossy fibres --- p.13 / Chapter 1.4.3.4 --- Climbing fibres --- p.13 / Chapter 1.4.3.5 --- Deep cerebellar nuclei --- p.13 / Chapter 1.4.3.6 --- Other cerebellar neurons --- p.14 / Chapter 1.4.3.7 --- Neuroglia of the cerebellum --- p.16 / Chapter 1.4.4 --- Circuitry of the cerebellum --- p.17 / Chapter 1.5 --- Development of the Cerebellum --- p.21 / Chapter 1.5.1 --- Anatomical changes during cerebellar development --- p.21 / Chapter 1.5.2 --- Molecular control of cerebellar development --- p.25 / Chapter 1.5.2.1 --- Specification of the cerebellar region --- p.25 / Chapter 1.5.2.2 --- Neurogenesis from the ventricular zone --- p.26 / Chapter 1.5.2.3 --- Neurogenesis from rhombic lip --- p.29 / Chapter 1.6 --- Scope of the Thesis --- p.33 / Chapter CHAPTER 2 --- Materials and General Methods / Chapter 2.1 --- Materials for Molecular Biological Work --- p.35 / Chapter 2.1.1 --- Enzymes --- p.35 / Chapter 2.1.2 --- Chemicals and others --- p.35 / Chapter 2.1.3 --- Plasmid vectors and plasmid --- p.36 / Chapter 2.1.4 --- Solutions and media --- p.36 / Chapter 2.2 --- Materials for Tissue/Cell Culture --- p.38 / Chapter 2.2.1 --- Chemicals --- p.38 / Chapter 2.2.2 --- Culture media and solutions --- p.38 / Chapter 2.2.3 --- Culture cells --- p.39 / Chapter 2.3 --- Animals --- p.40 / Chapter 2.4 --- Materials for Immunocytochemistry --- p.40 / Chapter 2.5 --- Oligonucleotide Primers --- p.41 / Chapter 2.6 --- RNA Extraction --- p.44 / Chapter 2.7 --- Generation of cDNA from mRNA --- p.44 / Chapter 2.8 --- Preparation of Recombinant Plasmid DNA --- p.45 / Chapter 2.8.1 --- Small scale preparation of DNA --- p.45 / Chapter 2.8.2 --- QLAGEN plasmid midiprep kit method --- p.46 / Chapter 2.9 --- Preparation of Specific DNA Fragment from Agarose Gel --- p.46 / Chapter 2.10 --- Subcloning of DNA Fragments --- p.47 / Chapter 2.10.1 --- Preparation of cloning vectors --- p.47 / Chapter 2.10.2 --- Subcloning of DNA fragment --- p.48 / Chapter 2.10.3 --- Transformation of DNA into competent cells --- p.48 / Chapter 2.11 --- Preparation of Competent Cells --- p.48 / Chapter CHAPTER 3 --- Generation and Characterization of Induced Neurons / Chapter 3.1 --- Introduction --- p.50 / Chapter 3.2 --- Experimental Procedures --- p.51 / Chapter 3.2.1 --- Construction of expression vector --- p.51 / Chapter 3.2.1.1 --- Preparation of insert DNA --- p.51 / Chapter 3.2.1.2 --- Construction of entry vector --- p.52 / Chapter 3.2.1.3 --- Construction of destination vector --- p.52 / Chapter 3.2.1.4 --- Construction of expression vector --- p.52 / Chapter 3.2.2 --- Generation of induced neural cells --- p.57 / Chapter 3.2.2.1 --- Culture of mouse embryonic fibroblasts (MEF) --- p.57 / Chapter 3.2.2.2 --- Production of expression vector containing retroviruses --- p.57 / Chapter 3.2.2.3 --- Transfection and induction of neural fate of MEF --- p.57 / Chapter 3.2.3 --- Immunocytochemcial analysis --- p.58 / Chapter 3.2.4 --- Efficiency calculation --- p.59 / Chapter 3.3 --- Results --- p.62 / Chapter 3.3.1 --- A screen for cerebellar Purkinje and granule cell fate-inducing factors --- p.62 / Chapter 3.3.2 --- Characterization of the induced neurons --- p.67 / Chapter 3.3.2.1 --- Granule cell induction --- p.67 / Chapter 3.3.2.2 --- Purkinje cell induction --- p.71 / Chapter 3.4 --- Discussion --- p.102 / Chapter 3.4.1 --- Roles of inducing factors in Purkinje cells and granule cells development --- p.102 / Chapter 3.4.2 --- Mechanism of neural transdifferentiation --- p.107 / Chapter CHAPTER 4 --- Future Directions / Chapter 4.1 --- Complete Induction of Purkinje Cell Fate --- p.111 / Chapter 4.2 --- Induced Neurons of Different Subtypes --- p.112 / Chapter 4.3 --- Mechanism of Transdifferentiation --- p.114 / Chapter 4.4 --- Transdifferentiation and Regenerative Medicine --- p.114 / Bibliography --- p.116

Thesis (Ph. D.)--University of Virginia, 2007. / Title from title page. [alpha] in title is the Greek character. Includes bibliographical references. Also available online through Digital Dissertations.

Oral submucous fibrosis (OSF) is a debilitating irreversible fibrotic condition of the oral cavity. It is characterized by inflammation and ultimately results in trismus. Patients face difficulty in speaking, swallowing and chewing due to restricted mouth opening (trismus). This disease is also categorized as an oral premalignant disorder (OPMD). Recent reports cite a conversion rate of 10% from OSF to oral squamous cell carcinoma (OSCC). Epidemiological studies and case reports over the years have correlated the habit of chewing areca nut (Areca catechu) to the manifestation of OSF. It is a major cause of concern in the South and South East Asian parts of the world where areca nut is cultivated and routinely consumed. There are an estimated 700 million areca nut chewers around the globe with 0.5% of the population in the Indian subcontinent being affected by OSF due to this habit. Previous studies have reported differential gene expression profile and up regulation of the pro-fibrotic transforming growth factor-β (TGF-β) pathway in OSF. However, detailed molecular mechanisms for the pathogenesis of this disease are still unclear despite our knowledge about the etiological agent (areca nut) responsible for its progression. Therefore, to gain insights into the etiopathogeneses of OSF, following objectives were undertaken:  To study the gene expression changes induced by areca nut and pro-fibrotic cytokine TGF-β in primary fibroblast cells, and their implications in OSF.  To elucidate the mechanism of TGF-β signal activation in epithelial cells by areca nut. Fibroblast cells are the effectors in all fibrotic disorders. Therefore, it is essential to study the response of this cell type in fibrosis. With prior knowledge of the activation of TGF-β pathway in OSF and the etiological agent of this disease being areca nut; we wanted to study the differential gene response of fibroblasts to these two agents. For this purpose, human primary gingival fibroblasts (hGF) were used as a model system to study the global gene expression profile regulated by areca nut and/or TGF-β. hGF cells were treated with sub-cytotoxic dose of areca nut (5 µg/ml) with and without TGF-β (5 ng/ml) for 72 hours and microarray was performed. The results revealed 4666 genes being differentially regulated by areca nut in hGF cells while TGF-β regulated 1214 genes. Both of them together differentially regulated 5752 genes. 413 genes which were commonly regulated by areca nut and TGF-β were observed to have enhanced regulation with a combined treatment of areca nut, together with TGF-β. This result pointed towards the potential role of both areca nut and TGF-β in modulating fibroblast response. To further assess the role of areca nut in OSF manifestation, we first compared the transcriptome profile induced by it in epithelial cells with fibroblast cells. Areca nut was found to induce differential response in these two cell types which corroborates with the disease pathology wherein; epithelial atrophy is observed and conversely fibroblasts are proliferative. To extend these observations we compared the areca nut induced profile in epithelial cells with OSF differential profile and found that a majority of the genes regulated by areca nut which were common with OSF are regulated by TGF-β. Similarly, areca nut and TGF-β regulated profile in fibroblast cells overlapped significantly with OSF profile. Additionally, areca nut and TGF-β treatment positively enriched matrix associated and metabolic pathways among others which are reported to be differentially regulated in OSF. These observations also highlighted the importance of combined actions of areca nut and TGF-β in OSF manifestation. To test the physiological importance of combined actions of areca nut and TGF-β in the context of OSF; activation of fibroblasts by these treatments was assessed. Treatment of fibroblasts with areca nut and TGF-β enhanced the expression of myofibroblast markers αSMA and γSMA with a concomitant increase in the contractile property when compared to areca nut or TGF-β treatment alone. Further, we observed that areca nut did not regulate any of the TGF-β ligands or receptors in fibroblasts, whereas it induced TGF-β2 in epithelial cells. Therefore, this invoked a possible epithelial-mesenchymal interaction that may exist in OSF pathogenesis. To test this possibility in-vitro, epithelial cells were treated with areca nut and the secretome of these cells was put on hGF cells to study the regulation of fibrosis associated genes. This treatment enhanced the regulation of fibroblast activation markers (αSMA and γSMA) as compared to direct areca nut treatment. This increase in regulation was abrogated when induction of TGF-β2 was compromised in epithelial cells. Similar results were obtained for the regulation of other genes (TGM-2, THBS-1, EDN1, LOXL3, PLOD2, TMEPAI, TGFBI, CTGF, BMP1, LMIK1). Therefore, we concluded that TGF-β which is secreted in response to areca nut by epithelial cells influences fibroblasts in combination with areca nut to enhance fibrosis response. Furthermore, the secretome of untreated epithelial cells was found to down regulate the basal expression of fibrosis related genes in fibroblasts, invoking a role for epithelial secretome in regulating the fibrosis progression. Our data highlighted the importance of TGF-β’s influence on fibroblast response in OSF, but the mechanism for the regulation of this cytokine was not known. Areca nut did not induce TGF-β ligands in fibroblast as discussed above, but previous data from our group had reported areca nut mediated up regulation of TGF-β2 in epithelial cells. Therefore, we further elucidated the mechanistic details for this induction using immortalized keratinocytes (HaCaT and HPL1D) and correlated these in OSF tissues. The kinetics of the induction of TGF-β signaling by areca nut (5 µg/ml) in epithelial cells was established. Areca nut induced TGF-β2 transcript, protein and activated the canonical signaling (pSMAD2/3) at 2 hours post treatment, which persisted till 24 hours. The regulation of TGF-β2 mRNA at 2 hours was dependent on active transcription but was independent of protein translation whereas the activation of signaling (pSMAD2) required both transcription and translation at this time point. This warranted probing for the role of TβR-I in the activation of TGF-β signal by areca nut. A small molecule inhibitor was used to abrogate the kinase activity of TβR-I. Areca nut induced TGF-β2 mRNA at 2 hours even in the presence of TβR-I inhibitor whereas the induction was compromised at 24 hours although the activation of SMAD2 at both 2 and 24 hours was compromised in the presence of TβR-I. This result signified that induction of TGF-β signaling was dependent on the TβR-I activity at early and late time points, but the transcription of the ligand was independent of the receptor activity at early time point. These results indicated the activation of some other pathway by areca nut which could regulate the transcription of TGF-β2 and thereby activate TGF-β signaling in epithelial cells. To explore this possibility, a panel of pathway inhibitors was used and only JNK inhibitor compromised areca nut induced TGF-β2 mRNA and pSMAD2. The results were corroborated by transient knockdown of JNK1 and JNK2. Further, JNK was phosphorylated at 30 minutes to 2 hours by areca nut treatment on epithelial cells. This activation was found to be independent of TβR-I activity. In good correlation, activated JNK1/2 was also detected in OSF tissues and was not detectable in normal tissues. Since JNK activation was found to be a pre-requisite for areca nut induced TGF-β signal activation; we further explored the mechanism of JNK activation by areca nut itself. Areca nut mediated activation of JNK was found to be dependent on muscarinic acid receptor, Ca2+/CAMKII and ROS. Inhibition of these significantly compromised areca nut induced pJNK. In line with this, inhibition of muscarinic acid receptor activity, CAMKII and ROS also abrogated areca nut mediated induction of TGF-β2 mRNA and pSMAD2. The regulation of TGF-β signaling by areca nut in epithelial cells was dependent on transcription, and JNK activity was essential for this. We further sought to explore transcription factors which were regulated by JNK and therefore could possibly induce TGF-β2 promoter activity. ATF2 and c-Jun transcription factors were found to be induced at 30 minutes by areca nut and this up regulation also persisted till 24 hours. Further, activation of both ATF2 and c-Jun was dependent on JNK but independent of TβR-I activity. Moreover, areca nut treatment induced translocation of these phoshorylated transcription factors in the nucleus of epithelial cells. Additionally, pATF2 and p-c-Jun were enriched on TGF-β2 promoter after 2 hours of treatment by areca nut. To investigate the importance of this enrichment and regulation of TGF-β signal activation by areca nut, we transiently knocked down these proteins and studied the regulation of TGF-β2. Areca nut induced TGF-β2 mRNA and pSMAD2 was abrogated upon ATF2 and c-Jun knockdown, implicating JNK mediated activation of ATF2 and c-Jun in areca nut induced TGF- β signaling. To explore the significance of this mechanism in OSF, immunohistochemical staining for pATF2 and p-c-Jun was performed on OSF and normal tissues. Both the transcription factors were found in the nuclei of OSF tissues whereas their expression was not detected in normal tissues. This expression also correlated with the previously reported activation of SMAD2 in OSF tissues by our group. Hence, ATF2 and c-Jun were observed to be important in areca nut mediated TGF-β signaling in OSF. In conclusion, the work described in this thesis provides mechanistic details into OSF etiopathogenesis. Combined actions of areca nut and TGF-β induced a response in fibroblasts akin to OSF. Our results advocate a role for epithelial secreted factors in influencing fibroblast response in both normal and disease (OSF) conditions. Further, importance of TGF-β in OSF has been elucidated in terms of enhancing the fibroblast response to areca nut. We have also elucidated the mechanism for areca nut mediated activation of TGF-β signaling and have identified the contribution of JNK/ATF2/Jun axis in this process. This work can impact the management of oral submucous fibrosis by providing newer targets for treatment.