Mesenchymal-epithelial interactions are pivotal for proper pancreatic growth and development. The pancreatic progenitor cells present in the early pancreatic anlagen proliferate and eventually give rise to all pancreatic cell types. The Fibroblast Growth Factor 2b (FGFR2b) high-affinity ligand Fibroblast Growth Factor 10 (FGF10) has been linked to pancreatic epithelial cell proliferation and we have previously shown that Notch signalling controls pancreatic cell differentiation via lateral inhibition. By overexpressing FGF10 under the control of the Ipf1/Pdx1 promoter in mice, we have shown that persistent FGF10 activation in the embryonic pancreas of transgenic mice perturbs pancreatic epithelial cell proliferation and also inhibits pancreatic cell differentiation by maintaining Notch activation. In the Ipf1/Fgf10 transgenic mice, the pancreatic epithelial cells are ‘locked’ in an undifferentiated progenitor-like state with sustained proliferative capacity. Collectively, our data suggest a key role for FGFR2b/FGF10 signalling in the regulation of pancreatic growth and differentiation and that FGFR2b/FGF10 signalling interact with the Notch signalling pathway. Glucose homeostasis in mammals is critically dependent on co-ordinated glucose uptake, oxidative metabolism and insulin secretion in β-cells. Although, several key genes controlling various aspects of glucose sensing, glucose metabolism, insulin expression and secretion have been identified, we know relatively little about the molecular mechanisms that induce and maintain the expression of genes required for glucose-stimulated insulin secretion (GSIS) in β-cells. Attenuation of FGFR1c signalling leads to diabetes in mice. Overexpression of FGF2, a high-affinity FGFR1c ligand, under the control of the Ipf1/Pdx1 promoter also leads to diabetes in mice. The Ipf1/Fgf2 mice present with normal endocrine and exocrine differentiation but display impaired glucose-stimulated insulin secretion (GSIS), perturbed expression of genes required for glucose sensing uptake together with oxidative metabolism and increased expression of the FGF-signalling inhibitors Spry-2 and Pyst1/MKP3 in β-cells. Thus, stringent control of FGF signalling activation appears crucial for the maintenance of the regulatory circuit that ensures proper GSIS in pancreatic β-cells and hence normoglycaemia. The Wnt family of ligands via their receptors Frizzled (Frz) have been shown to mediate mesenchymal-epithelial interactions and cell proliferation in a variety of different systems. Expression of a plethora of Wnt ligands and Frz receptors has been previously reported in the pancreas and mice missexpressing Wnt1 and Wnt5a under the Ipf1/Pdx1 promoter display severely perturbed development. Here, we show the temporal and spatial expression of Wnt4, Wnt7b and Frz3 at different stages of pancreas development. To elucidate the role of Wnt signalling in the pancreas, we overexpressed a dominant negative form of mouse Frz8 under the Ipf1/Pdx1 promoter in mice. The Ipf1/Frz8CRD mice display severe pancreatic hypoplasia demonstrating that attenuation of Wnt signalling in the pancreas leads to perturbed pancreatic growth. Nevertheless, the transgenic mice present with normal endocrine and exocrine differentiation and remain normoglycaemic. The maintenance of normoglycaemia in these mice appears to be the consequence of a relative increase in endocrine cell number per pancreatic area combined with enhanced insulin biosynthesis and insulin secretion. Collectively our data provide evidence that Wnt signalling is required pancreatic growth but not adult β-cell function.

The teneurin C-terminal associated peptides (TCAP) are a family of four predicted peptides that are expressed in all metazoans where the teneurins have been studied to date. Of the four peptides, TCAP-1 has been studied most extensively. In vitro, TCAP-1 increases neuronal proliferation and neurite outgrowth. In vivo, the peptide reduces CRF-induced behavioural responses in rats. Despite the large body of evidence indicating a strong biological role for TCAP-1, little is known about the chemistry and solubility of the peptide, or the signaling pathway(s) mediating these effects. The aim of this research was to appropriately solubilize the peptide and to develop detection assays for its study in greater detail. I have now established an appropriate formulation of TCAP-1 and developed an immunoassay to assess its concentrations in tissues and in circulation. Also, by examining a number of transcriptional response elements, I have found two assays for probing the signal transduction mechanisms of this peptide.

The teneurin C-terminal associated peptides (TCAP) are a family of four predicted peptides that are expressed in all metazoans where the teneurins have been studied to date. Of the four peptides, TCAP-1 has been studied most extensively. In vitro, TCAP-1 increases neuronal proliferation and neurite outgrowth. In vivo, the peptide reduces CRF-induced behavioural responses in rats. Despite the large body of evidence indicating a strong biological role for TCAP-1, little is known about the chemistry and solubility of the peptide, or the signaling pathway(s) mediating these effects. The aim of this research was to appropriately solubilize the peptide and to develop detection assays for its study in greater detail. I have now established an appropriate formulation of TCAP-1 and developed an immunoassay to assess its concentrations in tissues and in circulation. Also, by examining a number of transcriptional response elements, I have found two assays for probing the signal transduction mechanisms of this peptide.

The epidermal growth factor receptor (EGFR) is a transmembrane receptor tyrosine kinase integral in regulating cell growth, survival, and migration. EGFR signaling, which is dependent on localization of the receptor within lipid rafts, is often hijacked during colon tumorigenesis. Previous work has found that docosahexaenoic acid (DHA) is protective against colon cancer. This fatty acid is proposed to function in part by perturbing lipid rafts and thereby altering cell signaling. The overall objective of this work was to determine whether DHA alters EGFR function and signaling. We assessed EGFR localization and ligand-induced phosphorylation in YAMC cells treated with fatty acids. We found that DHA reduced the localization of EGFR to lipid rafts. Concomitant with altering receptor localization, DHA was found to increase EGFR phosphorylation. However, DHA paradoxically suppressed EGFR signal transduction. We found that DHA uniquely altered EGFR activity, and other long chain polyunsaturated fatty acid did not exert the same effect. We additionally observed similar effects on EGFR activation and signaling by feeding mice a diet enriched in fish oil (high in DHA), and this was attendant with reduced colon tumorigenesis. We next probed the mechanism by which DHA enhances EGFR phosphorylation. We found that DHA facilitates receptor dimerization to increase phosphorylation. We additionally identified Ras activation as the site of perturbation of signal transduction. DHA suppressed signal transduction by both changing the localization of EGFR within the plasma membrane and increasing receptor endocytosis and degradation. Lastly, we extended our observations into a wounding model. Although DHA uniquely altered ligand-stimulated EGFR activity, both DHA and EPA altered EGFR transactivation and signaling upon injury. This culminated in reduced wound healing in DHA and EPA treated cells. In an animal model, we found that diets enriched in either DHA or EPA altered EGFR signaling in the colonocytes of wounded animals. Overall, we found that DHA modifies EGFR signaling, which can be beneficial or detrimental for health depending on the disease state of an individual. These data help elucidate a mechanism by which DHA protects against colon cancer, as well as indicating a potential downside of n-3 PUFA therapy.

Membrane rafts are highly dynamic heterogeneous sterol- and sphingolipid-rich micro-domains on cell surfaces. They are generally believed to provide residency for cell surface molecules (e.g., adhesion and signaling molecules) and scaffolding to facilitate the functions of these molecules such as membrane trafficking, receptor transport, cell signaling, and endocytosis. Using laser scanning confocal microscopy and reflection interference microscopy (RIM), we studied the spatial and temporal distributions of membrane rafts and surface receptors, signaling molecules, and cell organelles during the formation of phagocytic contact areas. K562 cells, which naturally express CD32A, a cell surface receptor for the Fc portion of Immuno-globulin g (IgG), was chosen as a model for neutrophils. An opsonized target was modeled using a glass supported lipid bilayer reconstituted with IgG. CD32A was found to cluster and co-localize with membrane rafts. Placing the K562 cells on the lipid bilayer triggered a process of contact area formation that includes binding between receptors and ligands, their recruitment to the contact area, a concurrent membrane raft movement to and concentration in the contact area, and transport of CD32A, IgG, and membrane rafts to the Golgi complex. Characterization of these processes was performed using agents known to disrupt detergent resistant membranes (DRMs), dissolve actin microfilaments, and inhibit myosin motor activity, which abolished the CD32A clusters and prevented the contact area formation. The relevance to phagocytosis of contact area formation between K562 cells and lipid bilayers was demonstrated using micro-beads coated with a lipid bilayer reconstituted with IgG as the opsonized target instead of the glass supported planar lipid bilayer. Disruption of membrane rafts, salvation of the actin cytoskeleton, and inhibition of myosin II activity were found to inhibit phagocytosis. Here we have provided evidence that membrane rafts serve as platforms that are used to pre-cluster CD32A and transport CD32A along the actin cytoskeleton to the site of phagocytic synapse formation, followed by internalization to the Golgi complex.

This research project presents a comprehensive functional analysis of a complex prokaryotic signal transduction system and the mechanisms underlying its evolution. The chemotaxis system regulates motility in prokaryotes and is their most complex signal transduction system. The system has been extensively characterized experimentally, but recent studies have created new questions about the function and origin of this system. Comparative genomics analyses are well-suited for studying the chemotaxis system since it is present in taxonomically diverse organisms. The first aim of this project is to understand the evolutionary history of the chemotaxis system that has resulted in the diversity of chemotaxis systems that have been experimentally. The results reveal three functional families of chemotaxis systems that regulate flagellar motility, type IV pili motility, and non-motility outputs. The flagellar family shows extensive diversity with 10 conserved classes that have variable accessory proteins, and these classes show a co-evolutionary relationship with flagella. The second aim of this project is to analyze the molecular evolution of chemotaxis system components and utilize that information to predict the contact sites involved in protein-protein interactions. The analysis supports that there is evolutionary pressure at the amino acid sequence level to maintain protein-protein interactions. From this observation, a method to predict the contact sites of protein-protein interactions from sequence information alone was developed and validated by experimental and structural information.

The effect of steady shear stress on somatic embryos were investigated in a flow chamber and evaluated at different time intervals using microscopy technique. The development of meristematic cell clusters, i.e. the immature embryos, into a polarized somatic embryo, and the effect on the localization of the suspensor cells that form during development of the immature embryos, were studied as a function of shear stresses. With the distribution and growth rate of the meristematic and suspensor cells, the effect of stress on the embryo development was established. Furthermore, the effect of shear stress on the cells at molecular level, the reaction of integrin-like proteins, the production of reactive oxygen species and the pore size of the cell walls involved in the shear stress responses, were investigated with molecular techniques. In general, shear stress inhibits meristematic cells growth. Meristematic cells grow fastest at shear rate of 86 s-1 among all the tested shear stress conditions. By combining the results of meristematic cells growth and suspensor cells formation, it suggests that there is a critical shear rate between 86 and 140 s-1, at which no suspensor cells form. The unidirectional flow with different shear stresses helps the polarized growth and the unidirectional alignment of suspensor cells. Reactive oxygen species and integrin-like protein are detected in the stressed cells as cellular responses to shear stresses. By monitoring the pore size and uptake time of cells to macromolecules with solute-exclusive experiments, it suggests that the stressed cells expedite the response to plasmolyzing components that are used to induce maturation treatment thus affect the response to maturation stimuli.

Calcium ion plays a pivotal role as second messenger during signal/response coupling in plant cells (Trewavas, 1999). Elevations of cytosolic Ca2+ occur in plants as a consequence of abiotic and biotic stresses, environmental and hormonal stimuli. However, the molecular mechanism by which changes in cytosolic calcium are sensed and transduced in the plant cell has not been completely elucidated. The detection of Ca2+-binding proteins, especially Ca2+-dependent protein kinases (CDPKs) in plants led to drawing analogy with animal systems wherein the Ca2+-message is perceived and transduced by proteins that bind Ca2+. CDPKs are stimulated by the direct binding of Ca2+ to their endogenous calmodulin (CaM) -like domain (Harper et al, 1991). CDPKs exist as multiple isoforms in a single species, and show tissue-specific and developmentally regulated expression. Furthermore, the diversity among different CDPK isoforms with respect to Ca2+-binding properties, activation, substrate specificity, regulatory mechanisms and other kinetic properties suggest their specialization in the regulation of distinct signaling pathways. These observations therefore have led to the speculation that most of the Ca2+-mediated signal transduction in plants occurs via the mediation of CDPKs (Harmon et al, 2000). Over the last 15 years there has been a dramatic unfolding of information on Ca2+-mediated signaling in plants. Nevertheless, little is known about the environmental/hormonal signals and the signaling events that regulate early plant developmental processes such as embryogenesis, seed development and germination. The present investigation was initiated with the objectives 1) to determine the role of Ca2+ during embryogenesis, 2) to examine the involvement of a CDPK during early developmental processes in sandalwood plant (Santalum album L.) and 3) to purify and biochemically characterize this CDPK. The study initially investigated the possible involvement of calcium-mediated signaling in the induction/regulation of somatic embryogenesis from proembryogenic cells of sandalwood. 45 Ca + uptake studies and fura-2 fluorescence ratio photometry were used to measure changes in [Ca2+]cyt of proembryogenic cells in response to culture conditions conducive for embryo development. Sandalwood proembryogenic cell masses (PEMs) were obtained in the callus proliferation medium that contains the auxin 2,4-D. Subculture of PEMs into the embryo differentiation medium which lacks 2,4-D and has higher osmoticum resulted in a 4-fold higher 45Ca2+ incorporation into the symplast. Fura-2 based ratiometric analysis also showed a 10-16- fold increase in the [Ca2+]cyt of PEMs under identical culture conditions, increasing from a resting concentration of 30-50 nM to 650-800 nM. Chelation of exogenous Ca2+ with EGTA arrested such an elevation in [Ca2+]cyt. Exogenous Ca2+ when chelated or deprived also arrested embryo development and inhibited the accumulation of a Ca2+-dependent protein kinase (swCDPK) in embryogenic cultures. However, such culture conditions did not cause cell death as the PEMs continued to proliferate to form larger cell clumps. Culture treatment with W7 reduced embryogenic frequency by 85%, indicating that blockage of Ca2+-mediated signaling pathway(s) involving swCDPK and/or CaM caused inhibition of embryogenesis. These observations suggest a second messenger role for exogenous Ca2+ and the existence of Ca2+-mediated signaling pathway(s) during sandalwood somatic embryogenesis. The detection of a 55 kD protein showing cross reactivity with polyclonal antisoybean CDPK and the detection of Ca2+-dependent protein kinase activity in protein extracts from somatic embryos, prompted investigation on the spatio-temporal accumulation and activity of a CDPK in different developmental stages of sandalwood. Western blot analysis and protein kinase assays identified a Ca2+-dependent protein kinase (swCDPK) of 55 kD in soluble protein extracts of different developmental stages of sandalwood somatic embryos. However, swCDPK was not detected in plantlets regenerated from somatic embryos. swCDPK exhibited differential expression and activity in the developmental stages of sandalwood. Zygotic embryos, endosperm and seedlings showed high accumulation of swCDPK. However, the enzyme was not detected in the soluble proteins of shoots and flowers of sandalwood tree. swCDPK exhibited a temporal pattern of expression in endosperm, showing high accumulation and activity in mature fruit and germinating stages, the enzyme being localized strongly in the storage bodies of the endosperm cells. Interestingly, these storage bodies were thereafter identified as oil bodies, suggesting that a Ca2+-mediated regulation of oil hydrolysis and/or mobilization might be operative during seed germination. swCDPK in the zygotic embryo was found to be inactive during seed dormancy and early stages of germination, indicating a possible post-translational hibition/inactivation of the enzyme during these stages. The temporal expression of swCDPK during somatic/zygotic embryogenesis, seed maturation and germination thus suggests involvement of the enzyme in these early developmental processes. In view of the diversity exhibited by members of the CDPK family, characterization of swCDPK, the early development specific CDPK from sandalwood was undertaken. Purification of swCDPK was achieved by chromatography on DEAE-cellulose, hydroxyapatite and Blue-Sepharose. The purified enzyme resolved into a single band on 10 % polyacrylamide gels, both under denaturing and non-denaturing conditions. swCDPK was strictly dependent on Ca2+, K0.5 (apparent binding constant) for Ca2+-activation of substrate phosphorylation activity being 0.7 μM and for autophosphorylation activity —50 nM. Ca2+-dependence for activation, CaM-independence, inhibition by CaM-antagonist (IC50 for W7 = 6 μM, for W5 = 46 μM) and cross-reaction with polyclonal antibodies directed against the CaM-like domain of soybean CDPK, confirmed the presence of an endogenous CaM-like domain in the purified enzyme. Kinetic studies revealed a Km value of 13 mg/mL for histone III-S and a Vmax of 0.1 nmolmin-1rng-1. The enzyme exhibited high specificity for ATP with a Km value of 10 nM. Titration with Ca2+ resulted in enhancement of the intrinsic emission fluorescence of swCDPK and a shift in the λmax emission from tryptophan residues. A reduction in the efficiency of non-radiative energy transfer from tyrosine to tryptophan residues was also observed. These are taken as evidence for the occurrence of Ca2+-induced conformational change in swCDPK. The emission spectral properties of swCDPK in conjunction with Ca2+ levels required for autophosphorylation and substrate phosphorylation help elucidate the possible mode of Ca2+ activation of this enzyme.

Transforming growth factor-β (TGF-β) is the proto-type member of a super family of secreted proteins comprised of several structurally related, but functionally divergent proteins like the BMP, activin, inhibin, mullerian inhibitory substance etc. TGF-β was originally identified as a secreted factor, which in the presence of EGF was capable of transforming normal rat kidney fibroblasts. Studies over the years have shown that this protein is multifunctional that influences several processes including development, immune function, epithelial cell growth and motility, wound healing etc. TGF-β plays important role in the normal physiology as well as in pathological conditions in mammals. There are three mammalian isoforms that are involved in several developmental processes as has been shown by the knockout mice models. An important role for TGF-β has been implicated in several disease processes like fibrotic disorders (of liver, lung, kidney), inflammatory disorders (rheumatoid arthritis), autoimmune disorders (systemic lupus erythematosus) and cancer. TGF-β has a dual role in carcinogenesis. Initially it acts as a tumor suppressor and causes growth arrest of epithelial cells and cells in the early stages of cancer. But in an established tumor, TGF-β exerts an effect which is favorable for the survival, progression and metastasis of the tumor by promoting epithelial-mesenchymal transition (EMT), angiogenesis and escape from immune surveillance. Studies using mouse models have shown that an intact TGF-β signaling is essential for the metastasis of breast cancer. These observations indicate that the normal epithelial cells show differential response to TGF-β as compared to the tumor they give rise to. Supporting this, it has been shown that prostate tumor cells show invasive behavior in response to TGF-β and not non-tumorigenic cells. Most actions of TGF-β are brought about by regulation of gene expression and differential gene expression mediated by TGF-β has been reported in tumor cells and normal cells. For example, in response to TGF-β, tumorcells show increase in the production of proteases like uPA, MMPs etc and down regulation of the inhibitors of proteases TIMP isoforms, whereas this is not observed in the normal cells. However, there is no clear understanding of the mechanism (s) responsible for differential responses of various cell types to TGF-β. Since a role for TGF-β has been established in several pathological conditions particularly cancer and fibortic disorders, this pathway are a very attractive target for therapeutic intervention. Hence, if the TGF-β pathway has to be targeted for therapy of any disease, it becomes essential to identify the targets of TGF-β in different cell-types and their mechanism of regulation, particularly in un-transformed and transformed cells. Over the past few years, there have been several independent transcriptome analyses of cells in response to TGF-β treatment in various cell types such as HaCaT, fibroblasts, corneal epithelial cells etc. From a comparison of these studies, it is noted that TGF-β regulates genes in a cell type specific manner. Considering the dual role of TGF-β on normal and transformed cells, identification of genes and/or biochemical pathways regulated by TGF-β in these cells may allow identification of therapeutic targets for diseases involving TGF-β signaling pathway. With this background, the following objectives were set for the current investigation: 1. Identification of targets of TGF-β in normal and tumor cells and also the genes differentially regulated by TGF-β 2. Understand the mechanism of regulation of a few selected genes 3. Characterize novel targets of TGF-β with respect to their regulation by TGF-β and also their function Towards the aim of identification of targets of TGF-β in different cell-lines, expression profiling of genes in response to TGF-β was performed in a lung adenocarcinoma cell line (A549) and a matched immortalized lung epithelial cell line (HPL1D). Our data showed similar regulation of 267 genes in HPL1D and A549 cells by TGF-β. This suggests that the genes commonly regulated in both HPL1D and A549 are not tumor specific. Some of these genes were also reported to be regulated by TGF-β in other studies using micro array in various cell types. While 1757 genes are exclusively regulated by TGF-β in A549, only 733 genes are exclusively regulated in HPL1D cells. The reasons for this differential response are not known. However, some of the genes exclusively regulated in A549 such as Integrin αV, thrombospondin 1 have been shown to aid tumor survival, maintenance and metastasis. In contrast, in HPL1D, TGF-β regulates tumor suppressor genes like WT1, ECM proteins like collagen which are responsible for arrest of cell growth and apoptosis. This differential gene regulation in normal and tumor cells may explain the dual role of TGF-β in carcinogenesis. The differences in the effects of TGF-β on these two cell-lines could be due to the phenotypic properties of these cells, HPL1D being a non-transformed cell-line and A549 being a transformed cell-line. It is also possible that the differences are due to cell-type specific effects. In order to address this question, expression profiling in response to TGF-β was carried out using another cell-line namely HaCaT, which is an immortalized skin keratinocyte cell-line. When the expression profiles of the three celllines namely HPL1D, HaCaT and A549 in response to TGF-β treatment were compared, it was found that the genes regulated by TGF-β can be divided into seven categories based on the cell-line in which they are regulated. In this comparison, it was seen that there were several genes which were regulated by TGF-β in A549 and HaCaT despite the fact that these two cell-lines have little in common. The reason for these two celllines to show similarities in their gene expression profile in response to TGF-β is unclear. When the genes regulated by TGF-β in the three cell-lines were categorized based on their annotated functions using the DAVID tool, it was found that signaling pathways like MAP kinas, focal adhesion, Wnt signaling are regulated by TGF-β in all the celllines. On the other hand, Integrin αV was found to be regulated in A549 and HaCaT cells and very marginal regulation was seen in HPL1D cells. This could be one of the reasons for the similarities between A549 and HaCaT. There are studies which show the role of Integrin αV in some of the TGF-β mediated actions although the mechanism by which Integrin signaling modulates gene expression is not well understood. Our data shows that indeed thrombospondin 1 which is regulated by TGF-β in A549 and HaCaT is regulated through the integrin signaling pathways as blocking this pathway partially blocks the induction of this gene by TGF-β. TGF-β actions on cells are to a large extent are carried out by the phosphorylation of SMAD 2/3 by activated TGF-β type I receptor upon TGF-β signaling. Several genes that are transcriptionally regulated by TGF-β contain a SMAD complex binding element (SBE). However, over the last few years, evidences have accumulated which suggest that some actions of TGF-β could be independent of SMADs, mediated by the other signaling pathways like the MAP kinas, PKC and others. In order to understand the mechanism of regulation of a few selected genes by TGF−β, inhibitors for the three MAP kinas pathways (p38, ERK and JNK) were used prior to treatment with TGF-β. The expression of these genes was assessed by qRT-PCR analyses. These studies showed that most of the genes regulated by TGF-β require one or more of the MAP kinas pathways. In HaCaT and A549, the number of genes dependent on the MAP kinas pathways is more compared to HPL1D. Based on our data, we propose that activated MAP kinas pathway could be one of the essential determining factors for the various differential actions of TGF-β in tumor cells. However, the reason for the behaviour of HaCaT cells, which are untransformed cells in a manner similar to the A549 cells, is still unclear. One of the reasons for the similarity could be the activation of the integrin signaling pathway as described before. The expression profiling data identified several novel targets of TGF-β. One such target is S100A2, a calcium binding protein containing an EF hand motif that has been implicated in cancer. A progressive reduction in the expression of this gene has been reported with increasing grade of the tumor. Our studies show that this gene is regulated by TGF-β in HaCaT and HPl1D, but not in A549 cells. The induction of S100A2 by TGF-β in HaCaT cells is likely to be transcriptional as it is sensitive to actinomycin treatment. We further investigated role of other signaling pathways in the regulation of S100A2 by TGF-β and found that the regulation of this gene by TGF-β depends on the ERK and also the integrin signaling pathways. In order to characterize this gene with respect to its functions, A549 cells were chosen as they have very low endogenous expression of S100A2. Hence, in order to explore if there is any role for the loss of S100A2 expression in the progression of A549 cells, we cloned the DNA of S100A2 in a mammalian expression vector, transected A549 cells with this and isolated clones stably expressing this gene. We performed assays to assess cell proliferation, cell migration and potential to form colonies in soft agar. The data suggests phenotypic differences in the colonies that formed in soft agar and no major differences in other assays. Overall, our data has identified several novel targets regulated by TGF-β other than S100A2 like IGFBP7, FGFR1, and SPUVE etc. Further, regulation of several genes was found to be in a cell type specific manner involving MAP kinase and integrin signaling pathways. This study also identified major differences in the genes regulated by TGF-β in transformed and non-transformed lung epithelial cells.

Glycodelin is a 162 amino acid dimeric, glycosylated, secretory protein of the lipocalin superfamily. Its classification as a lipocalin(carriers of small hydrophobic molecules) is based mainly on the presence of lipocalin signature motifs in its primary sequence and no ligand for this protein has been identified till date. Glycodelin has 40-55% sequence identity with β-lactoglobulin which is the second type member of the lipocalin superfamily (the first being retinol binding protein (RBP). Glycodelin is primarily a primate specific protein (though there have been isolated reports of mRNA in mice and rats) with many isoforms secreted by various tissues, predominantly of the reproductive tract. These isoforms, being the product of the same gene, are identical in primary sequence and differ only in their glycosylation due to differences in tissue origin; hence they may be better addressed as glycoforms of glycodelin. The main glycoforms of glycodelin reported till now are GdA, GdS, GdM, GdF and GdC. Each glycoform of the protein has a varied function, dictated or modulated largely by the complex glycans on its surface. GdA, the most well studied glycoform of glycodelin, is secreted by the endometrium under progesterone control and accumulates in the amniotic fluid (from where it is isolated.). GdA has been subclassifed as an immunocalin (immuno-modulatory lipocalins) due to the many immuno-modulatory functions pertaining to tissue differentiation, implantation and angiogenesis and most sifnificantly, modulation of immune responses at ehe feto-maternal interface. The fetus expresses paternal allo-antigens on its surface and would be regarded as foreign or non-self by the maternal immune system. Yet the fetus is not rejected, and is in fact protected from attack by the maternal immune system by a variety of tolerogenic mechanisms. GdA is the most abundant secretary glycoprotein of the primate uterine compartment during implantation and early pregnancy. It has been shown to have inhibitory effect on innate as well as adaptive and humoral immune responses. It inhibits the proliferation of T and B cells, Nk cytoxocity and suppresses monocyte chemotaxis. It also skews the cytokine profile from Th1 to Th2 and inhibits IL1 and IL2 secretion from mitogenically stimulated lymphocyte and mononuclear cell cultures. In our laboratory, we have demonstrated earlier that the inhibitory effect of GdA on T cell proliferation is due to apoptosis being induced. The apoptotic signaling induced by GdA was found to be caspase dependent and follows the intrinsic mitochondrial stress induced pathway of apoptosis. Having determined the effect of glycodelin A on T cells, we wanted to look at its effect on other cells playing a role in immune responses. We decided to look at its effect, if any, on the innate immune system. Chapter 1 of the thesis describes our studies on the effect of GdA on monocytes. We have looked at the effect of GdA on primary monocytes isolated from blood of healthy human volunteers and found that GdA induces apoptosis of primary monocytes and this appears to be mediated through a caspase independent pathway. The mitochondrial membrane potential of primary monocytes was lost upon GdA treatment therefore the mitochondria seem to be involved in the apoptotic cascade. As the yield of monocytes from peripheral blood is very low, further studies on the effect of GdA on monocytes were carried out using a human monocytic cell line, THP1, as a model system. We have demonstrated the GdA is able to inhibit the proliferation of these cells and also induce apoptosis in them. We also found that this signaling is partially caspase-dependent and involvement of other caspase independent pathways is possible. Further, we have shown that there is no effect of GdA on the phagocytic ability of these cells after differentiation into the macrophage lineage. However, when added before differentiation, glycodelin is able to inhibit the phagocytic ability of THP1 cells. We also found that THP1 cells were relatively resistant to GdA-induced apoptosis post differentiation into macrophages. We have also looked at the effect of GdA on B cells using primary B cells as well as a B cell line U266B1 as our model system. GdA was shown to inhibit the proliferation of primary B cells as well as of the cell line. The protein was not able to induce apoptosis in the primary cells (both activated as well as unactivated cells) as well as in the cell line. Treatment of the cells with MAP kinase inhibitors also did not render them susceptible to GdA induced apoptosis(as has been seen in the case of U937 cells). U266B1 cells remained relatively resistant to GdA-induced apoptosis even when treated for long periods. They did not undergo significant necrosis uponGdA treatment even though the proliferation of these cells was inhibited by the protein. We were surprised to find that there was loss of mitochondrial membrane potential of the cells upon GdA treatment even when there was no cell death. The reason for this is not clear. The inhibition of proliferation of B cells by GdA does not involve caspases and the signalilng induced by GdA in these cells seems to be different to that induced in T cells atleast downstream of the mitochondria as the cells cannot proliferate in presence of GdA but seem immune to further damage or apoptosis. These studies have been described in chapter 2 of the thesis. The third and final chapter of the thesis deals with our investigation into the effect of GdA on Nk cells. GdA, in an earlier report, has been shown to inhibit the activity of circulatory NK cells. However, the mechanism of this action has not been delineated. We made attempts to determine the effect of GdA on NK cells using a human NK cell line YT Indy as our model system, as isolation and culture of primary Nk cells in good numbers is difficult. Preliminary studies revealed that GdA triggered apoptosis in these cells. However, the process was found to be caspase independent. Another surprising finding was that GdA did not bring about significant loss of mitochondrial membrane potential of these cells, implying that the involvement of mitochondria in the apoptotic signaling in these cells may be at the later stages, as amplifiers rather than initiators, as has been seen in the case of T cells and monocytes.