Conselho Nacional de Desenvolvimento Científico e Tecnológico / Tem sido descrito que o acúmulo de mutações em proto-oncogenes e genes supressores de tumor contribui para o direcionamento da célula à carcinogênese. Na maioria dos casos de câncer, as células apresentam proliferação descontrolada devido a alterações na expressão e/ou mutações de ciclinas, quinases dependentes de ciclinas e/ou inibidores do ciclo celular. Os tumores sólidos figuram entre o tipo de câncer mais incidente no mundo, sendo a quimioterapia e/ou hormônio-terapia, radioterapia e cirurgia os tratamentos mais indicados para estes tipos de tumores. Entretanto, o tratamento quimioterápico apresenta diversos efeitos colaterais e muitas vezes é ineficaz. Portanto, a busca por novas moléculas capazes de conter a proliferação destas células e com baixa toxicidade para o organismo se faz necessário. Este trabalho teve por objetivo avaliar a ação antitumoral in vitro de um novo composto sintético, a pterocarpanoquinona LQB118, sobre algumas linhagens tumorais humanas de alta prevalência e estudar alguns dos seus mecanismos de ação. As linhagens tumorais estudadas neste trabalho foram os adenocarcinomas de mama (MCF7) e próstata (PC-3), e carcinoma de pulmão (A549). A citotoxicidade foi avaliada pelo ensaio do MTT e a proliferação celular pela contagem de células vivas (exclusão do corante azul de tripan) e análise do ciclo celular (citometria de fluxo). A expressão gênica foi avaliada por RT-PCR e a apoptose foi avaliada por condensação da cromatina (microscopia de fluorescência-DAPI), fragmentação de DNA (eletroforese) e marcação com anexina V (citometria de fluxo). Das linhagens tumorais testadas, a de próstata (PC3) foi a que se mostrou mais sensível ao LQB 118, e em função deste resultado, os demais experimentos foram realizados com esta linhagem tumoral. O efeito citotóxico do LQB 118 se mostrou tempo e concentração dependente. Esta substância inibiu a proliferação celular e prejudicou a progressão do ciclo celular, acumulando células nas fases S e G2/M. Buscando esclarecer os mecanismos desta ação antitumoral, demonstrou-se que o LQB 118 inibe a expressão do mRNA do fator de transcrição c-Myc e das ciclinas D1 e B1, e induz a apoptose de tais células tumorais. Em suma, o LQB 118 é capaz de inibir a proliferação das células tumorais de próstata, alterando a expressão do mRNA de alguns genes reguladores do ciclo celular, resultando em interrupção do ciclo celular e indução de apoptose, indicando este composto como um potencial candidato a futuro medicamento no tratamento do câncer de próstata. / It has been reported that accumulation of mutation on proto-oncogenes and tumor suppressor genes directs cells to carcinogenesis. In most described cancer, cells display uncontrolled proliferation due to altered expression or mutations of cyclins, cyclin-dependent kinases and cell cycle inhibitors. The solid tumors are the most common cancer type in world. Chemotherapy and/or hormone-therapy, radiotherapy and surgery are the suitable treatment for this disease. However, chemotherapy has been shown several side effects and often ineffective. Therefore, the search for new molecules with antitumoral activity low cytotoxicity is needed. The aim of this work was to evaluate the in vitro antitumoral effects of a new synthetic compound, the pterocarpanquinone LQB 118, on tumor cell lines of high prevalence in the world and to study some mechanisms of action. Tumor cell lines of breast (MCF7), lung (A549) and prostate (PC3) were cultivated at RPMI medium with 10% of serum fetal bovine. The cytotoxicity was evaluated by MTT assay and the cell proliferation by cell counting (trypan blue exclusion) and cell cycle analysis (flow cytometry). Apoptosis was evaluated by chromatin condensation (fluorescence microscopy with DAPI), DNA fragmentation (electrophoresis) and annexin-V and iodide propidium staining. Gene expression was studied by RT-PCR. As LQB 118 (5 g/ml) induced cytotoxic effect mainly on prostate tumor cells, further experiments were then performed only with this tumor cell line. The LQB 118 cytotoxic effects were time and concentration-dependent. Furthermore, this substance inhibited cell proliferation and promoted cell cycle arrest, increasing cell number in S and G2/M phases. Studying the mechanisms of the LQB 118 antitumoral action, it was demonstrated that this substance inhibited the mRNA expression of the transcription factor c-Myc, cyclin D1 and cyclin B1 and also induced apoptosis of PC3. Concluding, LQB 118 impairs prostate tumor cells proliferation due to altered mRNA expression of some cell cycle regulator genes, resulting in cell cycle arrest and apoptosis induction, suggesting this compound as a good candidate for a future drug in prostate cancer treatment.

O objetivo do projeto foi desenvolver linhagens celulares para um anticorpo terapêutico anti-hTNFα e comprovar sua funcionalidade. Os genes anti-hTNFα foram clonados em células CHO para seleção da população estável mista, demonstrando expressão de anticorpo com reconhecimento de hTNFα em estrutura tridimensional. A população de transfectantes de maior produtividade específica foi escolhida para geração de linhagem monoclonal utilizando a tecnologia robótica ClonePix FL. Não houve diferença estatística entre o anti-hTNFα purificado e o produto de referência na cinética de ligação ao TNFα e reconhecimento diferencial por FcγRs em ensaios por SPR O ensaio de atividade funcional mostrou que o anti-TNFα desenvolvido pôde neutralizar a citotoxicidade induzida em células L929 e inibir a expressão de ELAM-1 em HUVEC. Os resultados finais permitiram identificar os três melhores clones, estáveis por 60 gerações. A comparabilidade entre o anti-TNFα desenvolvido e a referência permite admiti-lo como não inferior, um dos requisitos para o desenvolvimento de biossimilar. / The aim of the project was to develop a therapeutic anti-hTNFα antibody and evaluate its functionality. The anti-hTNFα synthezised genes were cloned into CHO cells and stable pools were selected, producing antibodies able to hTNFα three-dimensional structure recognition. The stable pools displaying higher antibody yields were the source for the generation of monoclonal lineage by ClonePix FL robotic technology. The clones selection proceeded using different criteria as cell density, specific productivity, fed-batch performance, kinetics measured by surface plasmonic resonance, hTNFα binding through ELISA, western-blotting and SPR, FcγRs binding by SPR. Besides, a small number of clones was tested in functional assays by the impairment of cytotoxicity of hTNFα over L929 cells and the inhibition of ELAM-1 expression by HUVEC. The long term stability testing allowed to finally select 3 top clones, not inferior to adalimumab reference by the above criteria.

Die Krebsentstehung wurde vor rund 80 Jahren auf veränderten zellulären Energiestoffwechsel zurückgeführt. Diese Hypothese konnte bisher weder experimentell bewiesen noch widerlegt werden. Durch den Einsatz zweier Modellsysteme mit unterschiedlicher Expression des mitochondrialen Proteins Frataxin konnte in der vorliegenden Arbeit <br> gezeigt werden, dass der mitochondriale Energiestoffwechsel einen Einfluss auf die Tumorentstehung zu besitzen scheint. Eine Reduktion des mitochondrialen Energiestoffwechsels wurde durch die hepatozytenspezifische Ausschaltung des mitochondrialen Proteins Frataxin in Mäusen erreicht. Der durch das Cre-/loxP-Rekombinasesystem erreichte organspezifische Knock-out wurde auf Transkriptions- und Translationsebene nachgewiesen. Anhand verminderter Aconitaseaktivität, geringeren Sauerstoffverbrauches und reduzierten ATP-Gehaltes im Lebergewebe wurde ein signifikant verminderter Energiestoffwechsel dargestellt. Zwar entsprach die Genotypenverteilung in den Versuchsgruppen der erwarteten Mendelschen Verteilung, dennoch war die mittlere Lebenserwartung der <br> Knock-out-Tiere mit ca. 30 Wochen stark reduziert. Bereits in jungem Alter war bei diesen Tieren die Ausbildung von präneoplastischen Herden zu beobachten. Mit proteinbiochemischen Nachweistechniken konnte in Lebergewebe 4-8 Wochen alter Tiere eine verstärkte Aktivierung des Apoptosesignalweges (Cytochrom C im Zytosol, <br> verstärkte Expression von Bax) sowie eine Modulation stressassoziierter Proteine (geringere Phosphorylierungsrate p38-MAPK, vermehrte Expression HSP-25, verminderte Expression HSP-70) aufgezeigt werden. Im inversen Ansatz wurde eine Steigerung des mitochondrialen Energiestoffwechsels durch stabile transgene Frataxinüberexpression in zwei Kolonkarzinomzelllinien erreicht. Diese Steigerung zeigte sich durch erhöhte Aconitaseaktivität, erhöhten Sauerstoffverbrauch, gesteigertes mitochondriales Membranpotenzial und erhöhten ATP-Gehalt in den Zellen. Die frataxinüberexprimierenden Zellen wuchsen signifikant langsamer als Kontrollzellen und zeigten im Soft-Agar-Assay und im Nacktmausmodell ein deutlich geringeres Potenzial zur Ausbildung von Kolonien bzw. Tumoren. Mittels Immunoblot war hier eine vermehrte Phosphorylierung der p38-MAPK festzustellen. <br> Die zusammenfassende Betrachtung beider Modelle zeigt, dass ein reduzierter mitochondrialer Energiestoffwechsel durch Regulation der p38-MAPK und apoptotischer Signalwege ein erhöhtes Krebsrisiko zu verursachen vermag. / Eigthy years ago, it was suggested that impaired energy metabolism might cause cancer. Compelling experimental evidence for this hypothesis is lacking. By use of two different model systems here we show that impaired expression of the mitochondrial protein frataxin leading to impaired mitochondrial energy metabolism appears to be <br> inversely related to tumour growth. To generate mice with reduced mitochondrial energy metabolism the expression of mitochondrial protein frataxin was disrupted in a hepatocyte-specific manner by using the cre/loxP-system. Presence, efficiency and specificity of disruption were shown at transcriptional and translational levels. Decreased activity of aconitase, reduced oxygen consumption and diminished ATP level in the liver revealed diminished energy <br> metabolism. Although knock-out mice were born in the expected Mendelian frequency, they exhibited a significantly decreased life expectancy. Young mice exhibited hepatic preneoplasia. The use of proteinbiochemical techniques revealed activation of apoptotic <br> pathways (cytochrome c in the cytosol, increased expression of bax) and modulation of stress-associated cascades (decreased phosphorylation of p38-MAPK, increased expression of HSP-25 and diminished expression of HSP-70). Inversely, transgenic overexpression of frataxin in colon cancer cell lines lead to increased mitochondrial energy metabolism as demonstrated by elevated activity of aconitase, increased oxygen consumption, elevated mitochondrial membrane potential and increased ATP levels. Frataxin-overexpressing colon cancer cells exhibit a <br> concurrent decrease in replication rate. The colony forming capacity in soft-agar-assay and tumour formation in nude mice were clearly decreased. Immunoblotting revealed elevated phosphorylation of p38-MAPK. Taken together, these models suggest that reduced mitochondrial energy metabolism may promote cancer through regulation of p38-MAPK and apoptotic pathways.

Interferons (IFNs) perform a wide range of biological activities: anti-microbial, anti-proliferative, immunomodulatory etc. The IFN family includes three main classes: Type I, Type II and the recently identified Type III. The two main members of Type I class are IFNα and IFNβ, which are well known for their anti-viral roles. IFNλ, a member of the Type III class of IFNs, also exhibits antiviral activity. IFNγ, also known as immune IFN, is a Type II IFN which is secreted, primarily, by activated T cells, NK cells and macrophages. IFNγ is a potent immunomodulator which plays important roles in host defense. The diverse functions of this cytokine are demonstrated in Ifnγ-/- mice which display increased sensitivity to several pathogens, high incidences of tumors, reduced inflammatory response etc. IFNγ binds to its cognate receptors, which consist of two subunits, IFNγ receptor (IFNGR) 1 and IFNGR2. IFNγ mediates its multifarious biological actions by activating the Janus activated kinase (Jak)-Signal transducer and activator of transcription (Stat) 1 signaling pathway. Jaks belong to a family of non-receptor protein tyrosine kinases and phosphorylate the IFNγ receptor and the transcriptional co-activator, Stat. IFNGR1, the larger subunit, is required for ligand binding and its carboxyl terminus is involved in binding to Jak1, which in turn phosphorylates Stat1. The smaller subunit, IFNGR2, is required for signaling and contains the Jak2 binding site. After binding of IFNγ to its receptor, a series of phosphorylation events occur, resulting in Stat1 phosphorylation and homodimerization of Stat1 to form the gamma activating factor (GAF). These activated molecules translocate to the nucleus and bind to gamma activating sequence (GAS) present in the promoters of several IFNγ-modulated genes. Thus, the cellular responses mediated by IFNγ are, primarily, due to modulation of gene expression. Therefore, the identification and study of IFNγ stimulated genes, signaling mediators and their cross talk with other cellular pathways is an active area of research. The system of our study was a hepatoma cell line, H6, which is derived from a spontaneous tumor from B10.A mice and selected for in vitro cell culture. It is an IFNγ inducible system and has been used to study IFNγ-induced gene expression and functional responses. Treatment of H6 cells with IFNγ greatly enhanced MHC class I levels but also reduced cell growth. High amounts of reactive oxygen species (ROS) and reactive nitrogen intermediates (RNI) play crucial roles in the growth suppressive effect of IFNγ. To better understand the signaling pathways involved in the generation of ROS and RNI, the involvement of Ras was investigated. Ras-GTP levels were determined by pull down assays using GST-Raf1-Ras binding domain fusion protein bound to glutathione agarose. Ras activation (conversion of Ras-GDP to Ras-GTP) was observed in H6 cells upon IFNγ treatment by ~12 hr. To assess the functional role of Ras activation, studies with Manumycin A, a farnesyl transferase inhibitor (FTI), were performed. The formation of functional Ras requires farnesylation, a post-translational modification, which is inhibited by FTIs. Treatment with Manumycin A blocked Ras activation but did not significantly modulate the IFNγ-induced MHC class I. However, the inhibitor reduced ROS amounts leading to increased cell growth in the presence of IFNγ. Together, these results delineated the role of Ras and ROS in modulating some functions of IFNγ. To further understand the mechanisms by which Ras mediates its functions during IFNγ mediated growth suppression, the activation and function of Ras effectors was evaluated. In particular, the role of Ras-like (Ral) guanyl nucleotide-binding proteins, RalA and RalB, was investigated. IFNγ induced transcripts of RalA but not RalB. Also, the induction of RalA and IFNγ induced growth suppression were Stat1-dependent. Studies involving chemical inhibitors and genetic studies revealed that Ras played a role in the induction of RalA during IFNγ treatment. The role of c-Jun N-terminal kinase (JNK), a stress induced kinase, was also elucidated in this system. Together, IFNγ induced activation of Ras and its effectors RalA and JNK, leading to high amounts of ROS that suppressed cell growth. To evaluate the physiological significance of Ras activation during inflammatory responses, the mouse model of acetaminophen (APAP) induced liver injury was established. Hepatotoxicity due to overdose of the analgesic and antipyretic, APAP, is a major cause of liver failure in adults. APAP is metabolized into a reactive metabolite which binds to glutathione. Consequently, the depletion of intracellular glutathione stores leads to oxidative stress and liver injury. Notably, Ifnγ-/- mice are resistant to APAP-induced liver damage demonstrating a crucial role for this cytokine. The role of Ras activation was evaluated after oral dosing of BALB/c mice with APAP. Ras-GTP was induced early and decreased amounts were observed upon treatment with L-methionine, which replenished glutathione amounts. Injection with L-methionine or Manumycin A rescued liver injury as assessed by lowered serum alanine aminotransferase amounts and histological analysis. Kinetic studies were also performed, under different treatment conditions, to estimate different biochemical parameters: glutathione amounts, JNK activation, protein carbonylation, ROS amounts, serum amounts of cytokines, TNFα and IFNγ etc. This study reveals a role of Ras activation in stimulating proinflammatory responses and demonstrates the therapeutic efficacy of FTIs during APAP-induced liver injury. In addition the role of RalA during APAP-induced liver injury was also studied. In summary, this study, involving in vitro cell culture and in vivo liver injury model systems, sheds light on the significant contributions of Ras and its effector, RalA, during IFNγ mediated growth suppression and APAP-induced liver injury.

Human endogenous retroviruses (HERVs) constitute about 8% of the human genome. There are around 4000 pol-containing retroviral integrations in the human genome, which makes it impractical to measure each of them separately. Therefore we developed a set of degenerate real time PCRs to detect major groups bearing sequence similarities to gammaretroviruses, one of the largest groups of human endogenous retrovirus, and betaretroviruses, some of which have integrated into the human genome most recently and which remain the most intact. It was found that, although both gammaretroviral and betaretroviral RNAs were broadly expressed in various healthy tissues including reproductive tissues and brain, a differential expression pattern was observed. My work further revealed that HERVE and HERVW, two gammaretroviral sequences, were ubiquitously and highly expressed in pathologic and normal female reproductive tissues with tissue specific patterns. Expression of HERVE was higher in endometriotic tissue than in normal endometrium. HERVE and HERVW RNAs were higher in normal ovarian tissue than in ovarian cancer. Besides these tissue- and neoplasia-related differences, there were wide differences in HERV expression among individuals. Next, a selective pattern of HERVW upregulation was demonstrated in SK-N-DZ, a neuroblastoma cell line, upon re-oxygenation after a period of hypoxia or with 5-azacytidine, a demethylating agent. Furthermore, broad and high expressions of gammaretrovirus-like transcripts in different brain areas analyzed were identified. The expression levels were variable among different donors. In conclusion a ubiquitous HERV expression was observed in tissues and cell lines, with various patterns. At this stage the data are not sufficient to conclude whether HERV has any physiological or pathological roles in humans. However, their differential expression patterns are compatible with functional roles of HERV in humans.

Since the publication of the complete sequence of the human genome in 2003 there has been great interest in exploring the functions of the proteins encoded by the genes. To reveal the function of each and every protein, investigation of protein localization at the subcellular level has become a central focus in this research area, since the localization and function of a protein is closely related. The objective of the studies presented in this doctoral thesis was to systematically explore the human proteome at the subcellular level using bioimaging and to develop techniques for validation of the results obtained. A common imaging technique for protein detection is immunofluorescence (IF), where antibodies are used to target proteins in fixated cells. A fixation protocol suitable for large-scale IF studies was developed and optimized to work for a broad set of proteins. As the technique relies on antibodies, validation of their specificity to the target protein is crucial. A platform based on siRNA gene silencing in combination with IF was set-up to evaluate antibody specificity by quantitative image analysis before and after suppression of its target protein. As a proof of concept, the platform was then used for validation of 75 antibodies, proving it to be applicable for validation of antibodies in a systematic manner. Because of the fixation, there is a common concern about how well IF data reflects the in vivo subcellular distribution of proteins. To address this, 500 proteins were tagged with green fluorescent protein (GFP) and used to compare protein localization results between IF to those achieved using GFP tagged proteins in live cells. It was concluded that protein localization data from fixated cells satisfactory represented the situation in vivo and together exhibit a powerful approach for confirming localizations of yet uncharacterized proteins. Finally, a global analysis based on IF data of approximately 20 % of the human proteome was performed, providing a first overview of the subcellular landscape in three different cell lines. It was found that the intracellular distribution of proteins is complex, with many proteins occurring in several organelles. The results also confirmed the close relationship between protein function and localization, which in a way further strengthens the accuracy of the IF approach for detection of proteins at the subcellular level. / <p>QC 20121017</p> / The Human Protein Atlas

Los cereales son empleados como dieta complementaria a la lactancia materna a partir del 4º mes ya que suministra nutrientes esenciales (especialmente hidratos de carbono, proteínas, minerales y vitaminas (particularmente tiamina). Los alimentos infantiles elaborados a partir de harinas procedentes de cereales pueden presentar compromiso en la biodisponibilidad de determinados minerales, por ello, es de gran importancia tratar de establecer los tratamientos tecnológicos necesarios para que la utilización de estos nutrientes esenciales sea la máxima. Se ha comprobado que el método más sencillo y eficaz de conseguir la eliminación del ácido fítico, es la adición de fitasa exógena. / Dietary minerals intake is of interest of human beings in general, but particularly for infant and young children in the first year of life, when growth is accelerated. Insuficient mineral intake in this period, mainly of iron, calcium and zinc, is responsible for diseases such as anaemia, rickets, osteoporosis or inmune diseases. Cereals are introduced to infants at the age of four to six months to supplement breastmilk and follow-on formula, since this is a period of rapid growth and development. Phytic acid (myoinositol hexa-phosphoric acid, IP6) is the major phosphorus storage compound of most seeds and cereal grains, and it has a strong ability to chelate multivalent metal ions, specially iron, zinc, and calcium.Based on this knowledge, complete phytate degradation by means of technological treatments is desirable, in order to overcome its negative effects on mineral bioavailability. Food processing-such as cooking, bread-making and fermentation-is known to reduce phytic acid content. However, the bioavailability of minerals can be considerably increased by dephytinization adding an exogenous phytase.

Genesis of life begins with the fusion of female and male haploid gametes through a process of fertilization leading to the formation of a diploid cell, the zygote. This undergoes successive cleavage divisions forming 2-, 4- and 8- cell embryos and their individual cells (blastomeres) are totipotent. As development proceeds, there is a gradual restriction in their totipotency, resulting in the generation of two distinct cell lineages i.e., the differentiated trophectoderm (TE) cells and the undifferentiated, inner cell mass (ICM) during blastocyst morphogenesis (Rossant and Tam 2009). During the course of development, the ICM cells can give rise to all cell types of an organism and can also provide embryonic stem (ES)-cells when cultured in vitro (Evan and Kaufman 1981). ES-cells are pluripotent cells, having the ability to self-renew indefinitely and differentiate into all the three primary germ layers (ectoderm, mesoderm and endoderm) derived-cell types. ES-cells are an excellent developmental model system to understand basic mechanisms of self-renewal, cell differentiation and function of various genes in vitro and in vivo (Capecchi 2001). Importantly, their cell derivatives could potentially be used for experimental cell-based therapy for a number of diseases. Although, human ES-cell lines have been successfully derived and differentiated to various cell types (Thomson et al., 1998; Odorico et al., 2001), their cell-therapeutic potential is far from being tested, in view of the lack of our understanding of lineage-specific differentiation, homing and structural-functional integration of differentiated cell types in the host environment. To understand these mechanisms, it is desirable to have fluorescently-marked ES-cells and their differentiated cell-types, which could facilitate experimental cell transplantation studies. In this regard, our laboratory has earlier generated enhanced green fluorescent protein (EGFP)-expressing FVB/N transgenic ‘green’ mouse, under the control of ubiquitous chicken -actin promoter (Devgan et al., 2003). This transgenic mouse has been an excellent source of intrinsically green fluorescent cell types. We have been attempting to derive ES-cell line from this transgenic mouse. Because the derivation of ES-cell line is genetic strain-dependent, with some strains being relatively permissible for ES-cell derivation while others are quite resistant (non permissive), it has been extremely difficult to derive ES-cell line from the FVB/N mouse strain. There is a need to evolve experimental strategies to derive ES-cell line from FVB/N mouse, a strain extensively used for transgenesis. Thus, the aims of the study described in the thesis are to: (1) develop an experimental system to derive EGFP-expressing fluorescently-marked ES-cell line from a non-permissive FVB/N mouse strain; (2) characterize the established ES-cell line; (3) achieve differentiation of various cell types from EGFP-expressing ES-cell line and (4) understand role of FGF signaling in cardiac differentiation from the established ES-cell line. In order to have an appropriate and relevant literature background, the 1st chapter in this thesis describes a comprehensive up-to-date review of literature, pertaining to the early mammalian development and differentiation of blastocyst, followed by origin and properties of ES-cells. Various ES-cell derivation strategies from genetically permissive and non-permissive mouse strains are described and also the ES-cell differentiation potential to various progenitors and differentiated cell types. Subsequently, details on molecular basis of cardiac differentiation and the therapeutic potential of ES-cell derived differentiated cell types to treat disease(s) are described. This chapter is followed by three data chapters (II-IV). Chapter-II describes the issues related to non-permissiveness of FVB/N strain for ES-cell derivation and strategies to overcome this hurdle. This is followed by detailed results pertaining to generation of homozygous EGFP-expressing transgenic mice and development of a two-pronged ES-cell derivation approach to successfully establish a permanent ES-cell line (named ‘GS-2’ ES-cell line) from the EGFP-transgenic ‘green’ mouse. This chapter also provides results pertaining to detailed characterization of the ‘GS-2’ ES-cell line which includes colony morphology, expansion efficiency, alkaline phosphatase staining, expression analysis of pluripotent markers by RT-PCR and immunostaining approaches and karyotyping. Following this, the outcome of results and significance in the context of reported information are discussed in detail. Having successfully derived the ‘GS-2’ ES-cell line, it is necessary to thoroughly assess the differentiation competence of the ‘GS-2’ ES-cell line. Therefore, the Chapter-III describes detailed assessment of the in vitro and in vivo differentiation potential of the ‘GS-2’ ES-cell line. For in vitro differentiation, results pertaining to ES-cell derived embryoid body (EB) formation and their differentiation to ectodermal, mesodermal and endodermal cell types, expressing nestin, BMP-4 and α-fetoprotein, respectively, are described. Besides, the robustness of adaptability of ‘GS-2’ ES-cells to various culture conditions for their maintenance and differentiation are described. Also shown in the chapter is the relatively greater propensity of this cell line to cardiac differentiation. For in vivo differentiation, the ‘GS-2’ ES-cell derived teratoma formation in nude mice and its detailed histological analysis showing three germ layer cell types and their derivatives are described. Last part of the data described in this chapter, pertains to generation of chimeric blastocysts by aggregation method. Because the ‘GS-2’ ES-cell line exhibited a robust differentiation potential, including an efficient cardiomyocyte differentiation, it is of interest to enhance the efficiency of cardiomyocyte differentiation by exogenous addition of one of the key growth factors i.e., FGF8b since this has been implicated to be critical for cardiogenesis in non-mammalian verterbrate species. Therefore, Chapter-IV is focused on assessing the ability of ‘GS-2’ ES-cell line for its cardiomyocyte differentiation property with particular emphasis on the FGF-induced cardiac differentiation. Results pertaining to the expressions of various FGF ligands and their receptors during differentiation of ES-cells are described. Besides, increases in the cardiac efficiency, following FGF8b treatment and the associated up-regulation of cardiac-specific markers such as GATA-4, ISL-1 and α-MHC are shown. At the end of data chapters, separate sections are devoted for ‘Summary and Conclusion’ and for ‘Bibliography’.