Yes / Polymer coatings that support epithelial cell culture have been developed. Ozonolysis and subsequent work up of poly(butyl methacrylate-co-butadiene) copolymers is used to form oligomers with carboxylic acid end groups, which are then further reacted with diamines to provide poly(butyl methacrylate)s with primary amine end groups. The polymers are cast as films and used as cell culture substrates for human dermal fibroblasts and human renal epithelial cells. Fibroblast and epithelial cells adhere and proliferate on acid functional materials but on amine functional films epithelial cells show greater viability than fibroblasts.

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.

Two Systems were designed. The first permits tunable on-demand softening of a hydrogel network. The second permits reversible on demand ligand exchange within a hydrogel network. Both means were shown to be cytocompatible and their uses demonstrated in cell culture of mesenchymal stem cells and 3T3 fibroblast cells.

The discovery that proprotein convertase subtilisin/kexin type 9 (PCSK9) mediates degradation of low-density lipoprotein receptors (LDLR) indicates a critical role in LDL metabolism. PCSK9 is a secreted protein that binds to the epidermal growth factor-like (EGF)-A domain of LDLR and directs the receptor for degradation in lysosomes by an unknown mechanism. A gain-of-function mutation, D374Y, increases binding to LDLR EGF-A >10-fold and is associated with a severe form of hypercholesterolemia in humans. Similar to previous studies, data obtained in my project has established that PCSK9 was capable of promoting robust LDLR degradation in liver-derived cell lines; however, minimal effects on LDLR levels were detected in several lines of fibroblast cells despite normal LDLR-dependent cellular uptake of PCSK9. Importantly, a PCSK9 degradation assay showed that 125I-labeled wild-type PCSK9 was internalized and degraded equally in both hepatic and fibroblast cells, indicating dissociation of wild-type PCSK9 from recycling LDLRs in fibroblasts. Moreover, PCSK9 recycling assays confirmed that no recycling of wild-type PCSK9 to the cell surface could be detected in fibroblast cells. In contrast, more than 60% of internalized PCSK9-D374Y recycled to the cell surface in these cells, and thus had reduced ability to direct the LDLR for lysosomal degradation despite persistent binding. Co-localization studies indicated that PCSK9-D374Y trafficked to both lysosomes and recycling compartments in fibroblast cells, whereas wild-type PCSK9 exclusively trafficked to lysosomes. We conclude that two factors diminish PCSK9 activity in fibroblast cells: i) an increased dissociation from the LDLR in early endosomal compartments, and ii) a decreased ability of bound PCSK9 to direct the LDLR to lysosomes for degradation. Finally, an LDLR variant that binds to PCSK9 in a Ca2+-independent manner could partially restore wild-type PCSK9 activity, but not PCSK9-D374Y activity, in fibroblast cells.

The discovery that proprotein convertase subtilisin/kexin type 9 (PCSK9) mediates degradation of low-density lipoprotein receptors (LDLR) indicates a critical role in LDL metabolism. PCSK9 is a secreted protein that binds to the epidermal growth factor-like (EGF)-A domain of LDLR and directs the receptor for degradation in lysosomes by an unknown mechanism. A gain-of-function mutation, D374Y, increases binding to LDLR EGF-A >10-fold and is associated with a severe form of hypercholesterolemia in humans. Similar to previous studies, data obtained in my project has established that PCSK9 was capable of promoting robust LDLR degradation in liver-derived cell lines; however, minimal effects on LDLR levels were detected in several lines of fibroblast cells despite normal LDLR-dependent cellular uptake of PCSK9. Importantly, a PCSK9 degradation assay showed that 125I-labeled wild-type PCSK9 was internalized and degraded equally in both hepatic and fibroblast cells, indicating dissociation of wild-type PCSK9 from recycling LDLRs in fibroblasts. Moreover, PCSK9 recycling assays confirmed that no recycling of wild-type PCSK9 to the cell surface could be detected in fibroblast cells. In contrast, more than 60% of internalized PCSK9-D374Y recycled to the cell surface in these cells, and thus had reduced ability to direct the LDLR for lysosomal degradation despite persistent binding. Co-localization studies indicated that PCSK9-D374Y trafficked to both lysosomes and recycling compartments in fibroblast cells, whereas wild-type PCSK9 exclusively trafficked to lysosomes. We conclude that two factors diminish PCSK9 activity in fibroblast cells: i) an increased dissociation from the LDLR in early endosomal compartments, and ii) a decreased ability of bound PCSK9 to direct the LDLR to lysosomes for degradation. Finally, an LDLR variant that binds to PCSK9 in a Ca2+-independent manner could partially restore wild-type PCSK9 activity, but not PCSK9-D374Y activity, in fibroblast cells.

Transforming growth factor-β (TGF-β) superfamily of cytokines comprises of several members, which can broadly be sub-divided into three classes [TGF-βs, Activin/Nodal, and Bone morphogenetic proteins (BMPs)]. Most members of this family play critical roles during embryo development differentiation and regulation of homeostasis. In mammals there are three TGF-β isoforms, TGF-β1, 2 and 3. All the three TGF-β isoforms have important roles in embryo development as revealed by mouse knock-out models. TGF-β has also been associated with several pathological conditions such as inflammation, Fibrosis, and cancer. In cancers, TGF-β plays both tumor suppressive and tumor promoting roles depending upon the context. TGF-β has growth inhibitory effect on epithelial cells which is essential to maintain tissue homeostasis. TGF-β induces the expression of several cyclin dependent kinase inhibitors such as p21Cip1, p15Ink4b while down-regulating the expression of cMYC in the epithelial cells. In lieu of its tumor suppressive role, several cancers harbor mutations in the components of the TGF-β signaling axis such as receptors and effector molecules called SMADs. Interestingly various cancers also show hyper activation of TGF-β signaling. It has been suggested that cancer cells become unresponsive to the growth inhibitory effects of TGF-β by losing the expression of p21Cip1, and p15INK4b. Oncogenic transformation of cancer cells can override the growth inhibitory effects of TGF-β. While the loss of growth inhibitory effects by TGF-β are seen in the tumor cells, several tumor promoting actions are also observed in these cells such as induction of EMT. TGF-β activates mesenchymal cells leading to the formation of a reactive stroma in tumors and TGF-β suppresses almost all types of cells of the immune system causing a local immune-suppressive environment. TGF-β also recruits mesenchymal stem cells into the stroma which secrete several cytokines. The sum total of all these effects is pro-angiogenic, pro-infiltrative and pro-metastatic. In the canonical TGF-β signaling pathway, ligands bind to the hetero-tetrameric receptor complex of TGFβR1 and TGFβR2 leading to activation of the TGFβR1 by TGFβR2. Activated TGFβR1 then phosphorylates and activates R-SMAD molecules (SMAD2, SMAD3) which complexes with the co-SMAD (SMAD4) and translocate into the nucleus to effect transcriptional changes. Non-canonical TGF-β signals are many and almost all the known signaling pathways like MAPK, WNT, PI3K-AKT, NOTCH, Integrin, Hedgehog, Hippo etc. have been shown to be activated by TGF-β in different contexts. The canonical TGF-β/SMAD pathway has been shown to be essential for both tumor suppressive and tumor promoting actions of TGF-β. Although the non-canonical signalling pathways have been shown to be context dependent, the exact mechanisms have not been elucidated. In previous studies, we have shown the importance of non-canonical TGF-β signaling in normal vs. carcinoma cells. However, there has been no study that addressed the differential effects of TGF-β on cells of connective tissue origin. To throw light on such questions we have undertaken this study with the following objectives: 1) Whole genome expression profiling of TGF-β targets in normal fibroblasts, transformed fibroblasts and sarcoma cells 2) Elucidation of non-canonical signaling pathways differentially regulated by TGF-β 3) Identification and characterization of novel TGF-β targets The cell-lines chosen for the study are: 1) hFhTERT (human foreskin fibroblasts immortalized with human terminal telomerase); 2) hFhTERT-LTgRAS (hFhTERT transformed with SV40 large T antigen and activated RAS); and 3) HT1080 (fibrosarcoma). We performed whole genome expression profiling using 4×44K Agilent Human Whole Genome Oligonucleotide Arrays. Analysis of the microarray results revealed that TGF-β regulated a large number of genes in all the three cell-lines but few targets were found to be commonly regulated between any two or all the three cell-lines. 5291 genes were differentially regulated by TGF-β between hFhTERT and hFhTERT-LTgRAS and 2274 genes were differentially regulated by TGF-β between hFhTERT and HT1080 cells. Gene set enrichment analysis (GSEA) of these two gene lists revealed enrichment of similar gene sets in the HT1080 and hFhTERT-LTgRAS cells compared to the hFhTERT cells. MAPK signaling pathway components were enriched in the hFhTERT cells. Closer inspection revealed that several upstream regulators of the MAPK pathway were in fact down-regulated by TGF-β in these cells compared to both hFhTERT-LTgRAS and HT1080 cells suggesting a depression of the MAPK pathway by TGF-β in the hFhTERT cells. Assessment of the phosphorylation status of ERK1/2 and p38 MAPK proteins after TGF-β treatment showed that both ERK1/2 and p38 MAPK pathways were not activated in response to TGF-β in the hFhTERT cells. On the other hand in hFhTERT-LTgRAS and HT1080 cells, both ERK1/2 and p38 MAPK were activated post TGF-β treatment. Activity of the AP1 and SMAD responsive p3TP-lux reporter plasmid was dependent on only the SMAD pathway in hFhTERT cells while in the hFhTERT-LTgRAS and HT1080 cells both MAPK and SMAD pathway were found to regulate the expression of the p3TP-lux reporter. This suggests activation of MAPK and SMAD pathways in transformed and tumor cells while there is no activation of MAPK in normal cells of mesenchymal origin. Components of the WNT signaling pathway such as WNT ligands WNT4, and WNT11, frizzled receptors, FZD4, FZD8 and FZD9, regulators like SFRP1, SFRP2, AXIN2 and several targets of the WNT-β-catenin pathway were regulated by TGF-β in the hFhTERT cells but not in the hFhTERT-LTgRAS and HT1080 cells suggesting a positive regulation of the pathway by TGF-β in the hFhTERT cells. Indeed, TGF-β induced the activity of the WNT responsive reporter, pTOP-FLASH in the hFhTERT cells but not in the hFhTERTLTgRAS and HT1080 cells. WNT4 and WNT11 were two of the novel targets of TGF-β identified in hFhTERT cells. Further experiments suggested that TGF-β conferred regulation of these genes was specific to the fibroblast cells since induction of these genes by TGF-β was not observed in any of the cancer cell lines or in HaCaT cells. Some recent studies have demonstrated remodelling of cytoskeleton in epithelial cells by the non-canonical WNT ligands such as WNT5a, WNT4 and WNT11. WNT4 has also been shown to be required for the maintenance of α-SMA levels in smooth muscle cells. In this study we have shown that WNT4 can induce α-SMA in the hFhTERT cells leading to their activation. TGF-β conferred activation of these cells was also found to be dependent on the presence of WNT4. In brief, our study identified differentially activated pathways by TGF-β in immortal and transformed fibroblasts. WNT4 was identified as a crucial molecule required for the TGF-β conferred activation of fibroblasts.

Indiana University-Purdue University Indianapolis (IUPUI) / Dynamic changes to the extracellular matrix (ECM) impact many cell fate pro- cesses. The ECM can experience changes in sti ness as well as changes in composi- tion in response to injury, development, and diseases. To better understand the role that these dynamic processes have on the cells residing within the environment, re- searchers have turned towards 4-dimensional (4D) hydrogel designs. These 4D hydro- gels re-capitulate not only 3-dimensional (3D) matrix architectures, but also temporal changes in the physicochemical properties. The goal of this thesis was to design a unify chemistry (i.e., Sortase A (SrtA)-mediated transpeptidation) for dynamic tun- ing hydrogel sti ness and the presence of bioactive ligands. The rst objective was to establish a tunable and cytocompatible enzymatic scheme for softening cell-laden hydrogels. Brie y, the e ects of SrtA-mediated matrix cleavage were investigated us- ing poly(ethylene glycol) (PEG)-peptide hydrogels crosslinked by SrtA-sensitive and insensitive peptides. Initially, the e ects of various parameters with respect to cat- alytic reactions of SrtA were characterized rheologically, including enzyme and sub- strate concentrations, macromer content, peptide composition, and treatment time. Gel moduli pre- and post-enzyme treatment were measured to verify SrtA-mediated hydrogel softening. The cytocompatibility of SrtA-mediated gel softening system was investigated using human mesenchymal stem cell (hMSC). Upon treatment with SrtA and an oligoglycine substrate, encapsulated hMSCs exhibited extensive spreading in comparison to those within statically sti matrices. The second objective was to es- tablish a reversible ligand exchange system utilizing SrtA-mediated transpeptidation. SrtA-sensitive pendant ligands were immobilized within PEG hydrogels, which were treated with SrtA and an oligoglycine substrate to a ord tunable removal of the pen- dant ligand. Through measurement of the liberated pendant peptide concentration, it was found that higher concentrations of SrtA or extending treatment times led to higher ligand removal e ciency. Finally, the e ect of peptide ligand removal on cell behaviors were evaluated using NIH 3T3 broblasts. Fibroblasts were culture both on and within hydrogels containing SrtA-cleavable cell adhesion peptide. After treatment, both conditions led to a decrease in broblast spreading in comparison to non-treated gels. Overall, the utility of SrtA as versatile agent for controlling the mechanical properties and the presence of biologically active components within a hydrogel system was demonstrated. These systems could be further explored with natural-based materials to better mimic the physiological environment experienced by cells.

No description available.

Orientador: Prof. Dr. Arnaldo Rodrigues dos Santos Junior / Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biotecnociência, 2014. / Na engenharia de tecidos, a utilização de uma fonte de celular juntamente com um biomaterial, representa uma alternativa clínica a ser aplicada a pacientes com graves lesões no tecido ósseo. Este estudo teve como objetivo avaliar células Vero, uma linhagem de célula fibroblásticas, e uma linhagem de células-tronco mesenquimais (Rat (SD) Mesenchymal Stem Cells) em testes de biocompatibilidade in vitro com polímeros de poli(hidroxibutirato-co-hidroxivalerato) (PHBV), poli(caprolactona) (PCL) e blendas 75/25 e 50/50 desenvolvidos para a bioengenharia de tecido ósseo. A diferenciação osteogênica das CTMs sobre os polímeros também foi analisada. Os biomateriais foram caracterizados morfologicamente através de Microscopia Eletrônica de Varredura (MEV), Estereoscópio e Micrômetro. Polímeros porosos se mostram mais espesso que os densos. Através das imagens obtidas nota-se a distribuição, tamanho e morfologia dos poros, notando que os polímeros se mostram com características condizentes a de outros trabalhos. As células Vero e células-tronco mesenquimais (CTMs) foram cultivadas sobre as amostras citadas. Realizamos o ensaio com o MTT, análise morfológica e citoquímica. Para as CTMs fizemos ainda ensaios para avaliar a diferenciação osteogênica (fosfatase alcalina e vermelho alizarina). Nenhum dos polímeros foi considerado tóxico para as células Vero e na maioria deles foi notada atividade celular, camadas de células bem distribuídas. As blendas 50/50 mostraram resultados um pouco inferiores, quanto ao MTT, essa blenda porosa demonstrou ser a uma amostra onde adesão ocorre de forma bem mais lenta, os demais polímeros apresentaram resultados semelhantes e até superiores ao controle positivo de adesão, principalmente o PCL denso. Apesar das amostras 50/50 densa e porosa não se mostrarem tóxicas, aparentemente não funcionam com bons substratos como os demais polímeros, também apresentaram várias dificuldades na técnica de preparação, sendo assim descartadas. A relação das CTMs com os biomateriais se mostrou semelhante aos resultados com Vero. As células foram capazes de se espalhar por quase toda a superfície dos polímeros inclusive entre os poros dos materiais porosos. As CTMs apresentaram resultados de adesão (MTT) sobre os polímeros, mais rápido do que a Vero, demonstrando também maior afinidade pelo PCL denso. Através da análise da atividade da enzima fosfatase alcalina (usada como marcador de diferenciação), notamos que as células-tronco se mostraram capazes de se diferenciar em contato com os polímeros. Esses dados foram confirmados com o vermelho de alizarina, que também mostrou que a diferenciação celular se mostra um pouco mais lenta nos materiais do que nas placas. De uma forma geral os polímeros com exceção das blendas 50/50 se mostraram como bons substratos para as células, com ausência de toxicidade, características morfológicas dentro do recomendado e, além disso, não bloqueiam respostas biológicas específicas, como a diferenciação osteogênica. / In tissue engineering, the use of a cell source coupled with biomaterial represents a clinical alternative which can be applied to patients with severe bone damage. This study aimed to evaluate the biocompatibility between Vero a fibroblastic cell line and Mesenquymal stem cells (Rat (SD) MSC) with biomaterials developed as scaffolds for bone tissue engineering, bioresorbable polymers composed of poly ( hydroxybutyrate-co-hydroxyvalerate ) [ PHVB ] and poly ( caprolactone ) [ PCL ] pure and ratios of (75 /25) and (50 /50) blends. The osteogenic differentiation of MSCs on the polymers was also analyzed. Morphological characterization of the materials was performed by Scanning Electron Microscopy (SEM), Stereoscope and micrometer. The porous polymers are thicker than dense. Through the images obtained it is possible to note distribution, morphology and pore size. The biomaterials seem to the same consistent characteristics of other studies. Vero cells and mesenchymal stem cells (MSCs) were cultured on the samples mentioned. We performed the assay with MTT, morphological and histochemical analysis. About MSCs we also evaluated the osteogenic differentiation (alkaline phosphatase and Alizarin red). None of the polymers was considered toxic to Vero cells and most of them presented cellular activity, layers of well-distributed cells was noted in most of them. However 50/50 blends showed no such significant results as other. In the MTT assay, this porous blend demonstrated to be the only sample where adhesion occurs more slowly, other polymers showed similar and even higher results than the adhesion positive control, especially the dense PCL. Although the dense and porous 50/50 samples do not show toxic, apparently they are not good substrates as the other polymers, also presented several difficulties in their preparation thus being discarded. The ratio of MSCs with biomaterials was similar to the Vero cells results. They were able to spread to almost all surface of the polymers including into the pores of porous materials. MSCs showed adhesion (MTT) faster than the Vero cell, also demonstrated a greater affinity for dense PCL sample. Through analysis of the enzyme alkaline phosphatase activity (used as a marker for differentiation), we noticed that the stem cells showed differentiation into contact with the polymers. These data were confirmed by alizarin red, which also showed that the cell differentiation was slower on the materials than on the plates. In general all the polymer blends but the 50/50 proved to be good substrates for cells, with no toxicity, morphological characteristics within recommended and in addition do not block specific biological responses, such as osteogenic differentiation.

Oral Submucous Fibrosis (OSF) is a chronic inflammatory disease resulting in progressive fibrosis of the oral tissues that can cause difficulty in chewing, swallowing, speaking, and mouth opening. Epidemiological studies have shown that OSF is a precancerous condition and 2-8% of the OSF patients develop squamous cell carcinoma. This disease affects 0.5% of the population in the Indian subcontinent and is now a growing public health issue in many parts of the world. Habit of chewing betel quid has been proposed as an important etiological factor in the development of this disease and is coline, a principle alkaloid of areca nut is considered as major causative factor for OSF development. But the exact molecular mechanism of OSF pathogenesis is not known. Therefore, we set the following objectives for this study: 1) Gene expression profiling of OSF using microarray. 2) Role of areca nut constituents in OSF pathogenesis. 3) Effect of areca nut on epithelial and fibroblast cells. In order to delineate the possible molecular mechanism of OSF pathogenesis, we took microarray approach and identified differentially regulated genes in ten OSF tissues against eight pooled normals using whole human genome oligonucleotide arrays. Microarray results revealed differential expression of 5288 genes (p≤0.05 and Fold change≥1.5), among them 2884 were up-regulated and 2404 were down-regulated. Validation employing quantitative real-time PCR and immunohistochemistry confirmed up-regulation of transforming growth factor-β1 (TGF-β1), TGFBI, THBS1, SPP1, TIG1 and down-regulation of bone morphogenic protein 7 (BMP7), C4orf7 and ALOX12 in OSF tissues. Furthermore, activation of TGF-β pathway was evident in OSF tissues as demonstrated by p-SMAD2 strong immunoreactivity. Analysis of IHC data showed that in all the normal tissues and in 70% of the OSF tissues the expression of TGF-β and BMP7 are inversely correlated. In good correlation, treatment of keratinocytes (HaCaT) by TGF-βdown-regulated BMP7, while BMP7 expression could not be detected in fibroblast cells. Hence, the imbalance between TGF-βand BMP7 signalling, which are positive and negative modulators of extracellular matrix production, respectively may trigger the manifestation of OSF. We also studied the regulation few genes (CTGF, TGM2 and THBS1) identified in OSF microarray in response to TGF-βand arecoline. TGF-βwas able to induce all the above genes in both HaCaT and hGF cells but arecoline could only induce TGM2 in hGF and THBS1 in HaCaT. Therefore TGF-βpathway came out to be the most important pathway in OSF microarray and subsequent validations. But areca nut constituents responsible for TGF-βpathway activation and the source (epithelial or fibroblast cells) through which it activates TGF-βare not known. In an attempt to understand the role of areca nut and its constituents in inducing TGF-βsignalling in epithelial cells, we performed microarray on epithelial cells (HaCaT) treated with areca nut water extract. Surprisingly, 64% of the differentially regulated genes by areca nut water extract matched with TGF-βinduced gene expression profile. To find out areca nut induced genes through TGF-β, epithelial cells were treated with areca nut in presence of ALK5 (TβRI) inhibitor. Out of 64% differentially induced genes, 57% genes induced by areca nut got compromised in presence of ALK5 and 7% were independently induced by areca nut, highlighting the effect of areca nut via TGF-β. Accordingly, areca nut treatment induced both p-SMAD2 and TGF-βdownstream targets TGFBI, TGM2, TMEPAI and THBS1 in HaCaT cells. One possible mechanism of TGF-βsignalling induction by areca nut could be via induced ligand (TGF-β2) and its activator (THBS1). Induction of TGF-β2 ligand by areca nut was shown at both RNA (Real Time) and protein (ELISA) levels. To find out areca nut components responsible for inducing TGF-β signalling, areca nut fractionation was performed which gave three fractions namely, Ethyl acetate (polyphenol), water supernatant (alkaloids) and Dichloromethane (impurity). Out of these; polyphenol and alkaloid fractions were found to be responsible for the induction of TGF-β signalling and its downstream targets. Upon treatment with purified components, catechin and tannin of polyphenol fraction and arecoline, arecaidine and guvacine of alkaloid fraction were found to be responsible for inducing TGF-β signalling, as seen by increased appearance of phopho-SMAD2 in HaCaT cells. Areca nut treatment on human gingival fibroblast cells (hGF) did not induce TGF-β signalling, highlighting that the source of TGF-β induction by areca nut could possibly be the epithelium. Further treatment of areca nut along with TGF-β on hGF cells potentiated TGF-β effect both in terms of TGF-β downstream targets like TGFBI, TGM2, TMEPAI, COL1A1 etc and activation of fibroblast by inducing α-SMA. Increasing concentration of areca nut is cytotoxic on HaCaT cells and pro-proliferative on hGF cells. This could provide a possible explanation for epithelial atrophy and proliferating fibroblast cells in connective tissue of OSF patients. Further exploration on HaCaT cell cytotoxicity by areca nut suggests the involvement of Reactive Oxygen Species (ROS) as a key molecule induced by areca nut. Compromising ROS generation by NAC (N-Acetyl-L-Cysteine) led to reversal of Sub-G1 peak induced by areca nut in HaCaT cells. This highlighted that cell death caused by areca nut could be ROS mediated. Areca nut treatment on hGF cells did not induce ROS generation, leading to no cytotoxicity on these cells. A possible explanation of this differential ROS generation can be due to dose dependent suppression of Catalase activity by areca nut in HaCaT cells but not in hGF cells. We also compared cytotoxicity of areca nut with all the alkaloids and found a good match with arecoline as both of them induce ROS, apoptotic ladder formation, annexin V positivity, suppression of Catalase activity and the cell death induced by them was compromised by NAC. The above results indicated that arecoline could be a mediator of areca nut water extract cytotoxicity on HaCaT cells. Betel nut chewer’s oral epithelium gets regularly exposed to areca nut and hence this exposure could be cytotoxic to oral epithelial cells too. We performed Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) in normal and OSF tissues. Our data showed 62.5% of OSF patients having significant percentage of epithelial cells with TUNEL positivity (Labeling index = 2-60%) compared to all normal tissues that were TUNEL negative. TUNEL positivity was predominantly seen in the upper keratin and supra basal layer of the epithelium. We also studied proliferation status of OSF epithelium and observed that 3-17% (LI) of epithelial cells in all normal tissues showed Ki-67 positivity in the germinal layer of epithelium. However, 65% of the OSF patients showed staining for Ki-67 (LI=.2-58%) in their epithelium. Also analysis of TUNEL positive and Ki-67 positive sections indicated that OSF patients with high TUNEL positivity have high Ki-67 labeling index, but stains in the supra basal or keratin layer (TUNEL) and basal layer (Ki-67) of epithelium respectively. This induced proliferation of epithelial cells could be the result of heavy apoptosis in the outer epithelium. But as these patients are regularly exposed to areca nut, this increased proliferation may not be able to cope up with the heavy apoptosis induced by areca nut, leading to atrophied epithelium. To understand the germinal status of OSF atrophied epithelium we performed staining for OCT4 in OSF tissues. To our surprise there were no OCT4 positive nuclei in the epithelium of 53% of OSF patients but a regular spread of OCT4 positivity has been seen in the epithelium of normal subjects. In conclusion, this thesis highlights the involvement of TGF-β pathway in OSF patho-physiology. In addition, activation of TGF-β pathway by areca nut constituents has been demonstrated. Moreover, the atrophied epithelium of OSF appears to be a consequence of apoptosis and stem cell deprivation. Taken together, areca nut perhaps causes atrophy of the epithelium and activates TGF-β pathway that may lead to manifestation of OSF.