Secretory phospholipase A2 (sPLA2) is an important part of apoptosis and disposal of damaged and dying cells. However, healthy cells are not susceptible to attack by sPLA2. Recent studies have focused on membrane properties necessary to induce susceptibility in both artificial and biological membranes. Hydrolysis of phospholipids by sPLA2 requires at least two preliminary steps: first, adsorption of the enzyme to the cellular membrane, and second, movement of a phospholipid into the active site of the enzyme. We determined the effects of susceptibility on each of the two steps and determined the contributions changing the equilibrium constants have on susceptibility. The equilibrium constant for step one increased by a factor of 2 during susceptibility, while the equilibrium constant for step two increased by a factor of 4. The rise in the second equilibrium constant caused the majority of the change in hydrolysis rate seen during susceptibility; the influence of the first equilibrium constant is minimal. We confirmed these results with adsorption studies (assessment of the first step). We additionally found that sPLA2 has a high affinity for the cellular membrane and that only a small percentage (3-5%) of the membrane is covered when all adsorption sites are filled by the enzyme. We proposed a mathematical model describing the mechanism of action of sPLA2, and we were able to experimentally justify the assumptions made in the model.

Secretory phospholipase A2 (sPLA2) behaves differently toward the membranes of healthy cells compared to those of damaged or dying cells. The enzyme catalyzes rapid and sustained hydrolysis of compromised cells consistent with a simple catalytic mechanism. In contrast, when healthy cells are incubated with sPLA2, they become resistant to hydrolytic attack as manifest by three unusual observations: First, hydrolysis is transient and represents only a small fraction of the total membrane phospholipid content. Second, subsequent addition of sPLA2 fails to generate additional product. Third, the apparent potency of the enzyme to cause the membrane to be refractory is much greater than the potency for catalyzing hydrolysis. The mechanism responsible for this resistance has not yet been identified. Using Monte Carlo and direct analytical methods, we have developed a model capable of explaining all three of these observations. The model requires two salient elements: only a small pool of phospholipids in the healthy cell membrane is available for catalysis by sPLA2, and hydrolyzed phospholipids are re-acylated and restored very slowly to the accessible pool. The requirement for initial hydrolysis (as opposed to the simple physical presence of the enzyme as previously thought) was confirmed experimentally. Additional evidence has shown that the membrane does not remain permanently in its resistant state. Over time, the membrane resets to its original state. The model also predicts that total substrate, reacylation rate, and the return rate of phospholipids to the membrane should all be constant as enzyme concentration is varied. This prediction was tested by quantitative analysis of hydrolysis time courses at varied enzyme concentrations. Experiments with fluorescent probes, merocyanine 540 and laurdan suggest, that resistance may also involve physical changes to the membrane beyond the kinetic mechanisms hypothesized in the model.

B cell development in chicken takes place in a specific primary lymphoid organ, the bursa of Fabricius. The bursa is considered to provide a microenvironment that promotes B lymphocyte survival and maturation. The most important maturation step in the bursa is the immunoglobulin (Ig) gene conversion, a process that is responsible for immunoglobulin repertoire in avian species. The Ig-gene conversion is strictly regulated, and only progenitors that are able to initiate the process will develop into fully functional B lymphocytes. In this study the late embryonic B lymphocyte stages are investigated, the bursal stem cell stage and the onset of Ig-gene conversion stage. Previous studies identified functional and phenotypic differences between the two stages, showing high rate of proliferation at both, but a significant increase in apoptotic activity at the onset of gene conversion stage. The molecular basis behind the initiation of Ig-gene conversion is not well understood. Here two approaches are presented to provide information on the B lymphocyte developmental process. In chapter II proteomic analysis of the two cell stages was performed. The proteins were sorted into functional groups and signal transductions pathways were identified that are associated with proliferation, differentiation, cell adhesion and apoptosis. The project identified differences in protein profiles that might explain the changes in B lymphocyte physiology and bursal microenvironment at the initiation of Ig-gene conversion. In chapter III the antigen recognized by a bursal secretory dendritic cell specific monoclonal antibody, GIIF3 was identified and cloned. The antigen was shown to be expressed by bursal secretory dendritic cells only during the late embryonic period. The antigen was identified as smooth muscle gamma actin. Futher work will investigate what role the gene plays in dendritic cell funtion.

No description available.

No description available.

No description available.

O fator de alcalinização rápida ou RALF (do inglês, Rapid ALkalinization Factor) é um peptídeo hormonal ubíquo que induz a atividade de uma MAP quinase, bem como um aumento rápido do pH extracelular do meio de cultura de células em suspensão. O AtRALF1, uma das 37 isoformas de RALF presentes em Arabidopsis thaliana, é um peptídeo secretado de 5 kDa, que inibe o crescimento radicular e causa uma rápida mobilização de Ca2+ extra e intracelular. Em células eucarióticas, calmodulinas são proteínas bem caracterizadas por mediar a transdução de sinais de Ca2+ intracelular. Entretanto, em várias espécies vegetais incluindo Arabidopsis, também existem relatos de calmodulinas secretadas com funções ainda desconhecidas. Neste trabalho, foi caracterizada a AtCML38, uma proteína de Arabidopsis semelhante a calmodulina e que interage com o peptídeo AtRALF1. Análises in silico e por RT-PCR semi-quantitativo mostram a co-expressão de ambos os genes AtRALF1 e AtCML38 em raízes de plântulas de Arabidopsis. Apesar da ausência de um peptídeo sinal típico na proteína AtCML38, a localização sub-celular demonstra que esta é secretada através da via secretória RE-Golgi. O uso de uma versão truncada da AtCML38, cujos 87 primeiros aminoácidos foram retirados, indicou que o N-terminal é essencial para o seu direcionamento. Por meio de ensaios de complementação fluorescente bimolecular, foi demonstrado que AtCML38 interage com AtRALF1 no apoplasto de folhas de tabaco. Ensaios de pulldown indicam que esta interação é específica e dependente de Ca2+ e do pH. Um mutante por inserção de T-DNA, que não produz a proteína AtCML38 (cml38), mostrou-se insensível ao peptídeo AtRALF1 aplicado exogenamente, e suas raízes cresceram normalmente. Plantas transgênicas de Arabidopsis super-expressando o gene AtRALF1 (35S:AtRALF1) têm um fenótipo semi-anão com pronunciada redução do crescimento radicular. Contudo, quando plantas 35S:AtRALF1 foram cruzadas com o mutante cml38, suas progênies exibiram fenótipo e crescimento radicular normais, apesar do acúmulo do peptídeo AtRALF1. Juntos, os resultados mostram que AtCML38 interage com o peptídeo hormonal AtRALF1, e que a proteína AtCML38 é essencial para inibição do crescimento radicular causado pelo peptídeo. / Rapid alkalinization factor (RALF) is a ubiquitous peptide hormone that induces a MAP kinase and a rapid increase in the pH of the extracellular media of cell suspension cultures. AtRALF1, one of the 37 RALF isoforms of Arabidopsis thaliana, is a secreted peptide of 5 kDa that inhibits root growth and causes a rapid mobilization of extra and intracellular Ca2+. In eukaryotic cells, calmodulin proteins are well-characterized to mediate intracellular Ca2+ signal transduction. Nevertheless, in several plant species including Arabidopsis, there are also reports of calmodulins being secreted with still unknown function. In this work, we characterized the AtCML38, a calmodulin-like protein from Arabidopsis as an AtRALF1-interacting protein. Analyses in silico and semiquantitative RT-PCR show co-expression of both AtCML38 and AtRALF1 genes in roots of Arabidopsis seedlings. Despite the fact that AtCML38 lacks a typical signal peptide, subcellular localization demonstrates that AtCML38 is secreted via the ER-Golgi secretory pathway. A truncated AtCML38, without the first 87 amino acids indicates that the N-terminal is essential for targeting. Through bimolecular fluorescence complementation assays, we showed that AtCML38 protein interacts with AtRALF1 in the apoplast of epidermal cells from tobacco leaves. Pull down assays indicate that this interaction is specific, Ca2+- and pH-dependent. A T-DNA insertion mutant defective in the AtCML38 protein (cml38) was insensitive to exogenously applied AtRALF1 peptide and their roots grow just as the wild type plants. Transgenic Arabidopsis plants overexpressing the AtRALF1 gene (35S:AtRALF1) have a semi-dwarf phenotype with a pronounced reduction in the root growth. However, when 35S:AtRALF1 plants are crossed with the mutant cml38, their progenies are normal looking and exhibit normal root growth in spite of the high accumulation of AtRALF1 peptide. Taken together, the results show that AtCML38 interacts with the peptide hormone AtRALF1 and that the protein AtCML38 is essential for the root growth inhibition caused by the peptide.

Apocynaceae presents the flowers with the highest degree of synorganization among the eudicots and highly elaborated pollination mechanisms associated with the high diversity of glands. The osmophore stands out as responsible to produce a floral scent which attracts pollinators and, despite its fundamental relevance for pollination, its structure and mechanism of production and release of the perfume is essentially unknown in Apocynaceae. This present work aims to characterize morphologically and ultrastructurally the osmophores of Apocynaceae, besides chemically identifying the compounds that constitute the floral scent. Species from two subfamilies were selected to describe the diversity of osmophores and types of scent in the family. The osmophores were firstly located histochemically and, later, this region was processed for transmission electron microscopy. Micromorphological analysis was performed by scanning electron microscopy, and the identification of volatile oils made by GC-MS. Osmophores are located on the adaxial surface of the free portion of the petals. They varied in the shape of epidermal cells, striation of the cuticle and presence of trichomes. This gland is mostly formed by secretory epidermis and parenchyma, except in Plumeria, where the osmophores are exclusively epidermal. The secretory cells presented thick walls in the Asclepiadoideae and secretion produced by plastids and SER in all species. Several vesicles perform the intercellular transport of secretion, as well as its release to the environment. However, Plumeria and Ditassa transfer the secretion produced to the vacuole before releasing it, and Tabernaemontana has a mixed release process. The composition of the scent varied among species, with great production of hydrocarbons, alcohols, ketones or monoterpenes depending on the species. Apocynaceae presents a high morphological and metabolic diversity in the osmophores which cannot be correlated with their morphology, subcellular organization, period of secretion release or pollination syndrome. This is the first comprehensive structural study of osmophores in this family that points out to very distinct evolutionary processes that may be related to multiple emergences in the phylogeny and speciesspecific associations with pollinators / Apocynaceae apresenta as flores com o maior grau de sinorganização das eudicotiledôneas e mecanismos de polinização altamente elaborados associados à mais alta diversidade de glândulas. O osmóforo destaca-se como responsável pela produção do perfume floral para atração dos polinizadores e, a despeito de sua importância fundamental para a polinização, a sua estrutura e mecanismo de produção e liberação do perfume é praticamente desconhecido nas Apocynaceae. O presente trabalho tem o propósito de caracterizar morfológica e ultraestruturalmente os osmóforos de Apocynaceae, além de identificar quimicamente os compostos que constituem o perfume floral. Espécies de duas subfamílias foram selecionadas, visando descrever a diversidade de osmóforos e tipos de perfume na família. Primeiramente, os osmóforos foram localizados histoquimicamente e, posteriormente, essa região foi processada para microscopia eletrônica de transmissão. Análise micromorfológica foi realizada através de microscopia eletrônica de transmissão. Análise micromorfológica foi realizada através de microscopia eletrônica de varredura e a identificação dos óleos voláteis, através de GC/MS. Os osmóforos estão localizados na superfície adaxial da porção livre das pétalas. Eles variaram quanto ao formato das células epidérmicas, ornamentação da cutícula e presença de tricomas. Esta glândula é, em sua maioria, formada por epiderme e parênquima secretores, exceto em Plumeria, onde os osmóforos são exclusivamente epidérmicos. As células secretoras apresentaram paredes espessas nas Asclepiadoideae e secreção produzida pelos plastídeos e REL em todas as espécies. Diversas vesículas realizam o transporte intercelular da secreção, assim como a sua liberação para o meio externo. Contudo, Plumeria e Ditassa transferem a secreção produzida para o vacúolo antes de liberá-la e Tabernaemontana possui processo de liberação misto. A composição do perfume variou entre as espécies, havendo grande produção de hidrocarbonetos, alcoóis, cetonas ou monoterpenos dependendo da espécie. Apocynaceae apresenta uma alta diversidade morfológica e metabólica em seus osmóforos que não pode ser correlacionada à morfologia, organização subcelular, período de liberação da secreção ou síndrome de polinização. Esse é o primeiro estudo estrutural abrangente sobre osmóforos na família e aponta para processos evolutivos muito distintos que podem estar relacionados a múltiplos surgimentos na filogenia e associações espécie-específicas com os polinizadores

O gene MAS1/GAS2, conhecido como \"Magnaporthe Apressoria Specific\", foi identificado como altamente expresso durante a formação do apressório do fungo filamentoso fito patogênico Magnaporthe grisea. Em Aspergillus fumigatus, fungo patogênico oportunista, o gene masA, ortólogo a MAS1/GAS2 foi identificado como upregulated em uma análise transcriptômica exposto a voriconazole e anidulafungina, antifúngicos que afetam a síntese do ergosterol e a parede celular, respectivamente. Em ambos os ortólogos apresentam uma estrutura na região N-terminal da proteína denominada peptídeo sinal o qual neste trabalho foi determinado em uma análise in silico a presença de peptídeo sinal na região N-terminal da proteína. Na caracterização fenotípica de uma linhagem masA - deletada de A. fumigatus não foi identificado alteração estrutural do conidióforo e no seu aspecto macromorfológico. A linhagem masA-deletada é resistente a voriconazol e farnesol, drogas que inibem a síntese de ergosterol e a uma molécula quorum sensing, respectivamente. Ao avaliar o nível de expressão gênica do masA frente a diferentes classes de drogas, foi observado que o gene esta super expresso quando ocorre dano na parede celular, membrana citoplasmática, DNA, inibições na síntese de lipídeos e ácidos graxos e no estresse oxidativo. Na presença de dano na parede celular fúngica causados por anidulafungina, a proteína MasA está localizado na parede celular próximo a ponta da hifa. Adicionalmente, foi capaz de transferir para o meio extra-celular a proteína fusionada a ele, a GFP. Assim, possivelmente MasA participa da via secretória do fungo principalmente em momentos de estresse como o desarranjo da parede celular. A capacidade de secreção natural dos fungos filamentosos tem sido explorada no contexto industrial há décadas. Indicando para este fim, uma possível aplicabilidade para este peptídeo sinal. / The MAS1/GAS2 gene, known as \"Magnaporthe Apressoria Specific\", was identified as highly expressed during the formation of the appressorium phyto pathogenic filamentous fungus Magnaporthe grisea. In Aspergillus fumigatus, opportunistic saprophytic fungus, masA gene orthologous to MAS1/GAS2 was identified as upregulated in a transcriptomic analysis exposed to voriconazole and anidulafungin, antifungals that affect the synthesis of ergosterol and the cell wall, respectively. In both orthologs have a structure at the N-terminus of the protein called signal peptide. During the phenotypic and functional characterization of masA gene in A. fumigatus, was determined on an in silico analysis the presence of signal peptide at the N-terminal region of the protein. In the phenotypic characterization of a strain masA - deleted, no structural change of conidiophores and its macromorfologic aspect was not identified. The characterization masA-deleted strain is resistant to voriconazol and farnesol drugs which inhibit ergosterol synthesis and a quorum sensing molecule, respectively. When evaluating the level of gene expression masA against different class of drugs was observed the super gene is expressed when damage occurs in the cell wall and membrane DNA, the synthesis of lipids and fatty acids, and oxidative stress. In the presence of damage in the fungal cell wall caused by anidulafungin, MasA protein is located near the cell wall and the tip of the hypha and additionally, was able to transfer the protein to the extracellular the protein fused to GFP. Thus, possibly MasA participates in the secretory pathway of the fungus especially in stress of the cell wall. The natural secretion capability of filamentous fungi has been exploited in the industrial context for decades. Indicating for this purpose, a possible applicability for this signal peptide.

Cellular differentiation is a complex but fascinating process in all multicellular organisms. Differentiation can involve changes in numerous aspects of cell physiology; size, shape, polarity, metabolic activity, responsiveness to signals, and changes in gene expression profiles. These changes form the basis for differentiation to occur. The human hemochorial placenta is an intricate apposition of fetal and maternal tissues that is strategically juxtaposed at the interface, with its widespread ‘villous’ or tree-like projections, directly in contact with maternal blood. It is therefore, ideally suited to perform life-sustaining functions such as exchange of nutrients, respiratory gases and metabolic wastes, with the maternal supply. It also plays a central role in the maintenance of the immunologically privileged status of the fetal semi-allograft. Placental development is directed towards the establishment of a continuous nutrient supply to the developing fetus. This requires efficient access of maternal blood to a transporting surface, the multinucleate syncytiotrophoblast layer. This is made possible by the rapid proliferation and ensuing invasion of mononuclear trophoblasts into the maternal uterus and remodeling of the spiral arteries therein. It is interesting to note that in early pregnancy, it is the placenta that first engages its active growth and proliferation and only then, permits the logarithmic growth phase of the embryo. As a developing organ, the placenta undergoes constant tissue remodeling, which is characterized by the functional loss of trophoblast cells by apoptosis. Most of these changes occur at the trophoblast layer of the placental villous that is composed of two cell types: cytotrophoblasts (CT) and syncytiotrophoblasts (ST). The mononuclear cytotrophoblast cells, which are located between the syncytiotrophoblast layer and its basement membrane, proliferate and fuse during trophoblast differentiation to form the overlying multinucleated syncytium. CT are highly proliferating and invasive cells, in contrast to the ST which are non proliferative less invasive and functionally very active. Syncytiotrophoblast cells form the continuous, uninterrupted, multinucleated, epithelium-like surface of the placental villous that separates maternal blood from the villous interior. ST performs a crucial role in feto-maternal exchanges and serves as an endocrine tissue by its ability to synthesize and secrete a variety of hormones such as GnRH, chorionic gonadotrophin (CG), placental lactogen (PL) and steroid hormones involved in the homeostasis during pregnancy. Thus, differentiation of CT into ST serves as an ideal model to study cellular differentiation as morphologically and functionally these cells exhibit highly contrasting features. The molecular basis of cytotrophoblast differentiation has been studied using primary cultures of human trophoblast cells as a model system. Highly purified preparations of mononucleated cytotrophoblast cells can be isolated from preterm and term placental tissue by enzymatic dispersion. The isolated cells from term placental tissue aggregate spontaneously in culture and fuse to form a multinucleated syncytiotrophoblast which synthesizes and secretes placental lactogen (hPL), chorionic gonadotropin (hCG) and other syncytiotrophoblast-specific protein and steroid hormones . These in vitro changes, which recapitulate important activities accomplished by normal cytotrophoblast cells during in vivo maturation, implicate a critical relationship between the differentiation of cytotrophoblast cells into syncytiotrophoblast cells. Though primary cell culture is an ideal model to study these changes, it comes inherently associated with various problems like health risk of handling human tissues, time involved, variability in each placental samples depending on health status of the subject and quite often lack of history of the subject which makes the results from these experiments difficult to reproduce and assess. One way to overcome this is the cell culture model which is a reproducible experimental system and permits the direct observation of time-dependent processes and their experimental manipulation. BeWo cells, the cells which we have used in our study, were derived from human gestational choriocarcinoma. These cells are the highly invasive malignant counterparts of the normal human trophoblast wherein, the limited capacity for cell proliferation is far exceeded. However, they still retain important features of their normal counterpart, like the potential of hormone production and induced differentiation. Differentiation of CT to ST is precisely controlled by different agents such as transcription factors, hormones, growth factors, cytokines and oxygen levels. BeWo cells have been used by other investigators as well as by us and it has been shown that these cells can be induced to differentiate with the agents mentioned above and terminally differentiate into cells which express typical characteristics of the normal differentiating trophoblast; like morphological transition from cytotrophoblast to syncytiotrophoblast-like cells, increased production of protein and steroid hormones (hCG, hPL, estrogens, progesterone); increased activity of cellular alkaline phosphatase and arrested cell proliferation. Since these cells can be triggered by external agents to differentiate, they serve as a useful model for the study of changes that occur during differentiation. Using primary cells and various cell lines including BeWo cells, various groups have attempted to study trophoblast differentiation and the regulators that control this process. The results of such study have only come out with a list of genes or proteins which might be having a role in this process and no functional correlation has been drawn so far from these studies. The members of the syncytin protein family, ADAM (a disintegrin and metalloprotease) proteins may well be some of the main players in the process of trophoblast fusion; some of the requisites of trophoblast fusion being redistribution of phosphatidylserine to the outer leaflet of the plasma membrane and activity of certain intracellular proteases. Clearly, further studies on trophoblast differentiation are needed to answer the question of the precise identity of regulatory proteins and role of these proteins during differentiation. The present study is aimed at gaining insights into the process of trophoblast differentiation and the molecular events which occur during this process. Our aim is also to study the regulated process of differentiation using BeWo cell model and identify the differentially expressed genes and relate the known function of these gene products to changes seen during differentiation process. We have employed the Differential Display Reverse Transcriptase Polymerase Chain Reaction (DD-RTPCR) and Microarray analysis to monitor the changes in gene expression. In CHAPTER 1, a brief account of morphological, biochemical and physiological changes which occur during placentation and trophoblast differentiation is discussed. Various aspects of placental function are discussed in brief, with special reference to the many unique abilities of trophoblast cells that contribute to a successful pregnancy. Detailed accounts of molecular mechanism of cellular differentiation, the models used in these studies and the advantages and drawbacks have been highlighted. The results of the previous studies from our laboratory using different model system and the outcome of the study are also outlined in this chapter. The advantages and disadvantages of the primary cell lines and the ease of handling of continuous cell culture model, BeWo is also presented in this chapter. The aim and objective of our study is to understand the molecular mechanisms underlying the trophoblast differentiation and the literature available is reviewed in the light of the objective and the aims and scope of the present study. The details regarding the materials used and the techniques employed during the entire study are outlined in CHAPTER 2-‘Materials and Methods’. The conditions for culture of BeWo human choriocarcinoma cell line are described and details of procedures employed for the validation of BeWo cells as a model system for monitoring the process of cellular differentiation are mentioned in this chapter. The details of the procedures employed for isolation of RNA, Reverse Transcriptase Polymerase Chain Reaction (RTPCR), Differential Display RT-PCR (DD-RT-PCR), Microarray analysis, Northern Blot analysis and Western Blot analysis are also described. The principle of the MTT assay used for verifying the viability of cells following various treatments is provided along with the working protocol. This chapter also includes protocols of the in vivo studies in rat, the methods employed for rat uterine mince cultures and isolation of rat uterine epithelial cells and dose and duration of the various treatments with steroid hormones and their inhibitors, treatment with protein kinase inhibitors in cell culture system are also described. In addition, this chapter also describes the procedures for transfection of hTERT, silencing of SLPI gene using SiRNA approach, gelatin zymography, MAP Kinase assay, FACS, cloning and expression of SLPI protein and procedure employed for raising antibodies to SLPI in rabbit. Finally, details of statistical tests employed fro anlaysis of data are presented. The results obtained in the present study are presented in 4 chapters(Chapters 3-6), CHAPTER 3 describes the characterization and validation of model system employed- BeWo cells to study human trophoblastic differentiation. BeWo cells under normal culture conditions resemble cytotrophoblasts like cells and when treated with various effectors of differentiation can be induced ot differentiate into syncytiotrophoblasts. We used 10 µM Forskolin to induce differentiation in BeWo cells. Forskollin is known to induce characteristic changes associated with human trophoblast differentiation in these cells. Incubation of BeWo cultures in the presence of 10 µM Forskolin resulted in dramatic morphological biochemical changes intheir cytotrophoblast-like phenotype. Mononuclear cells were seen to fuse to form multinucleate syncytial structures over a period of 72-96 hours in culture. This process was also associated with an increased production of β-hCG, Endoglin and hTERT thereby validating this model system for study of human trophoblastic differentiation. Analysis of cell cycle genes in this system established the arrest of proliferation thus further validating the system. The viability of these cells, during the entire period of culture, was verified using the MTT assay. This chapter discusses the importance of in vitro cell culture systems in the study of human placental development, and also addresses the suitability of these model systems for the study of human trophoblast proliferation and differentiation. One of the important finding of our earlier studies was that arrest of proliferation was a prerequisite for trophoblast differentiation to occur. This conclusion was based on the fact that telomerase expression which is a hallmark of all proliferating cells was down regulated in BeWo cells by 48h as assessed by TRAP (Telomere Repeat Amplification Protocol) assay or RT-PCR analysis for hTERT which is the catalytic subunit of telomerase. Telomerase activity was undetectable by about 96th by which time syncytium formation is normally completed after the addition of differentiation inducing agents like Forskolin, TGF β etc. Although the telomeric holo enzyme consists of many components the subunits which are critical for enzyme action are hTERT and hTR; hTR; hTR which is the RNA component of telomerase is ubiquitously expressed in most cell types including telomerase negative cells such as differentiated somatic cells. Since the BeWo cells can be induced to differentiate into multinucleated ST by addition of Forskolin and periodically the aged ST are eliminated by apoptosis. It is very well documented that the life span of ST is very limited and the ST have to be replaced by the freshly formed ST out of fusion of CT. Considering this, it was of interest to test whether differentiation can be prevented or delayed by extending the expression of telomerase activity. This would further validate our system that one of the requisites for cells to differentiate is down regulation of hTERT in BeWo cells. This was achieved by transfection of BeWo cells with hTERT expression vector. The results of the study clearly established that we were able to over express hTERT in BeWo cells; we also noticed an increase in the proliferation of BeWo cells as assessed by BrdU incorporation. In agreement with this observation is the fact that, in contrast to the empty vector transfected cells, in hTERT transfected group, the cell density appeared to be clearly more at 72 h. That the decrease in the hTERT expression in the control (empty vector transfected) is not due to cell death was established by MTT assay, which indicated that there was no difference in the viability between control and hTERT transfected cells. Further more, results of analysis for a variety of cell proliferation and differentiation markers by RT-PCR and Western blot analysis clearly supports the conclusion that hTERT over expression delays syncytium formation. Although reports are available on the differential expression of genes during differentiation of CT to ST with both primary cell lines as well as BeWo cell line, relatively less is known about the functional importance of differentially expressed genes. In CHAPTER 4, results of our studies to profile the differentially expressed genes during Forskolin induced differentiation in BeWo cells by two approaches DD-RTPCR and microarray analysis and relate the known functions of these genes to changes that occur during the differentiation of CT to ST are presented. We identified several genes that had robust change during differentiation by DD RTPCR and the differential expression of ten transcripts was confirmed by Northern blot analysis. The genes which we identified were SLPI, Elongation factor-1 alpha -1, Prolyl hydroxylase beta, LIMO-4 etc. These genes were either shown to have a role during differentiation of cells or have functional role in the syncytiotrophoblasts. Secretory Leucocyte Protease Inhibitor was one of the differentially expressed transcripts which were significantly up regulated during Forskolin induced differentiation of BeWo cells. SLPI which is a 12 KDa protein reported to exhibit a variety of activities which include inhibition of proteases and elastase, in addition to antibacterial and anti inflammatory activities. It was chosen for our further studies because of its multifunctional role in placenta and also during implantation. Micro array analysis revealed the up-regulation of hCG, hCS, and Endoglin thus validating the experimental system. Several candidate genes that could influence trophoblast differentiation, cell adhesion and cellular proliferation were identified. Genes involved in cellular proliferation include cyclin M3, replication factor 3, signal-induced proliferation-associated gene 1, osteonectin, clusterin, etc clearly indicating a growth-arrested phenotype for the differentiating BeWo cells. Trophoblastic differentiation associated genes included adipose differentiation-related protein, GADD45A, PPAR binding protein, galectin 3, tubulins, collagen, stathmin, etc. The p53 tumor suppressor protein plays a major role in cellular response to DNA damage and other genomic aberrations. Activation of p53 can lead to either cell cycle arrest or DNA repair or apoptosis. Although we did not observe any change in the p53 mRNA levels, the total protein level as well the phosphorylation status of p53 was up regulated upon differentiation. We confirmed the down regulation of Cyclin D1, D2 and PCNA in differentiated cells and up regulation of CDK inhibitors, P27kip1, P21cip1which are p53 induced genes by RT-PCR and Western blot analysis. Phosphorylation of ser 20 leads to reduced interaction of p53 with its negative regulator MDM2. MDM2 inhibits the accumulation of p53 by targeting it for ubiquitination and proteosomal degradation. Analysis for the phosphorylated status of p53 revealed that specifically the ser 20 phosphorylated p53, was increased upon differentiation. Phosphorylation of ser-392 has been reported to influence the growth repressor function, DNA binding and transcriptional activation of p53 and in agreement with this, western blot analysis revealed an increase in the ser-392 phosphorylated p53. These results suggest that p53, a nuclear protein regulating several genes involved in proliferation and differentiation is playing a pivotal role in growth arrest during trophoblast differentiation. We also noticed that several components of the apoptotic cascade are differentially expressed in cytotrophoblast and the syncytiotrophoblasts layer, and these changes appear to be associated with the stage of apoptosis. Apoptosis is involved in the removal of aging syncytiotrophoblasts and it also promotes cytotrophoblast fusion and formation of the syncytial layer. We found that various apoptosis related genes are up regulated and anti apoptotic genes suppressed following differentiation in our micro array analysis. We identified the involvement of p53 in this process also and chapter 4 deals with this aspect. Genes which regulate the invasive behaviour of trophoblasts which include MMP2, cathepsin K, cystatin N, SLPI and cysterine-rich angiogenic inducer 61, etc. were found to be up regulated following differentiation in our micro array analysis, which establishes these differences in gene expression reflects the physiological changes that occur during placentation. The Co-ordinated regulation of ptoteases and protease inhibitors I (for example SLPI, cystatin B and MMP2) suggests that these genes play an important role in the regulation trophoblast invasion at the uterine-placenta interface in vivo. Our studies revealed that one of the transcripts,namely, SLPI(Secretory leukocyte protease inhibitor) was robustly up regulated as assessed in DDRT-PCR, micro-array, Northern blot and RT-PCR analysis. Considering its importance in implantation, placentation and maintenance of pregnancy several aspects of this multifunctional protein were studied in detail and the results are presented in CHAPTER 5. Studies on the regulation of this transcript in Be-Wo cells revealed that SLPI mRNA is regulated by progesterone in Be-Wo cells. The up regulation of SLPI mRNA by progesterone was specifically inhibited by Progesterone receptor antagonist, RU 486 and estradiol 17β did not have any effect on the expression of SLPI mRNA expression in BeWo cells. The absence of regulation of SLPI by estradiol in BeWo cells was also established by the fact that simultaneous addition of progesterone and aromatase inhibitor, fadrazole did not block the increase in SLPI expression. Interestingly in vivo and in vitro studies using rat uterine minces and rat epithelial cells revealed that SLPI mRNA is regulated by Estradiol 17β and the effect is specifically inhibited by estrogen receptor antagonists such as ICI 182780, Tamoxifen, and Centchorman. Promoter analysis of rat and human SLPI revealed the absence of a consensus progesterone responsive element (PRE) in human and estrogen responsive element (ERE) in rat, suggesting the possibility of a non-genomic action of progesterone or estrogen in the induction of SLPI mRNA. This was confirmed by the observation that induction of SLPI mRNA could be effectively blocked by the addition of Staurosporine, an inhibitor of protein kinase C along with progesterone and estrogen to either BeWo cells or rat uterine epithelial cells. These results suggest that the non-genomic action of steroid hormones may be involved in the induction of SLPI. In the present study, we have also identified the intracellular signaling pathway that regulates SLPI gene expression by using various protein kinase inhibitors. We have also shown that activation of MAP kinase pathway upon progesterone treatment and the involvement of protein kinases in this activation, permitting us to conclude the non genomic action of progesterone in induction of SLPI mRNA in BeWo cells. The results of these studies are presented in detail in Chapter 5. The observation that SLPI expression is markedly increased during differentiation and differentially regulated by progesterone and estradiol, and induction by non genomic pathway prompted us to undertake studies to investigate its role during differentiation. This was accomplished by using SiRNA to silence the expression of SLPI in Forskolin induced differentiating BeWo cells and the results of this study are presented in CHAPTER 6. Different concentrations and combinations of oligos were used to silence the SLPI gene and we found that effective knockdown (>80%) was achieved with SiRNA concentrations ranging from 5-25nM. A combination of oligos also increased the knockdown from 50% to 90% as assessed by RT-PCR and western blot analysis for mRNA and protein levels of SLPI respectively. We found that inhibition of SLPI expression by SiRNA also inhibited the morphological differentiation of BeWo cells. Functionally this was reflected, by increase in the protease activity as assessed by gelatin zymography. It should be noted that SLPI is a protease inhibitor; it inhibits a variety of proteases, including proteases from neutrophils, pancreatic acinar cells and mast cells and SLPI present in the syncytiotrophoblast may have a crucial role in controlling protease activity associated with invasiveness and differentiation. Inhibition of differentiation by silencing the expression of SLPI provides an opportunity to monitor the changes in gene expression where in a single gene has been silenced in contrast to the model employed in chapter 4. We carried out microarray analysis using control (Forskolin treated) and SLPI silenced (Forskolin treated) samples. The results revealed that proliferation and differentiation, apoptosis and inflammatory pathways genes are affected due to SLPI silencing and the results of this study are presented in CHAPTER 7. We confirmed the changes in gene expression by semi quantitative RT-PCR analysis of the some important genes in each pathway. A comparison of the results obtained with that of our earlier microarray analysis which is described in chapter 4 revealed that the changes in levels of expression of the genes involved in cell proliferation, differentiation, apoptosis and inflammation were completely reversed after silencing the expression of SLPI. We have presented in chapter 5 the importance of MAP kinase pathway in Forskolin induced differentiation and the activation of this pathway when SLPI expression is increased following progesterone treatment. Interestingly after silencing the expression of SLPI we found that MAP kinase pathway is affected. It was observed that silencing of SLPI expression resulted in inhibition of activation of MAP kinase as assessed by the phosphorylation status by ELISA and no activation of MAP kinase was observed in SLPI silenced Forskolin treated cells. CHAPTER 8 provides a general discussion of the results obtained in the present study in the light of current understanding the type of genes involved, changes during human trophoblastic proliferation and differentiation and the key players during this process. This chapter also brings out the importance of SLPI during trophoblastic differentiation, placentation, implantation and its regulation by steroid hormones. The highlights and salient features of the present study are summarized in this chapter. In CONCLUSION, the present investigation has led to the identification of specific genes involved in trophoblast differentiation, human placental growth and development. Also evident from this study is the usefulness of the trophoblastic cell culture system for the study of cellular differentiation. We have attempted to relate the gene expression changes to physiological changes that occur during placentation, implantation and pregnancy. Many of the regulatory events that we have described during human trophoblastic differentiation, may not only be restricted to these cells, but may represent common principles/features of cellular differentiation in general. Loss of differentiation is a wide-spread feature of tumor progression, and frequently accompanies aggressive neoplastic behavior. Our studies provide unequivocal evidence to support cellular differentiation as a natural barrier to malignant transformation. Most importantly we have shown that silencing of a single gene can disrupt this differentiation process and the importance of SLPI during differentiation process perse.