Cette thèse est consacrée à l'étude de la topologie à l'infini d'espaces généralisant les graphes de Schreier. Plus précisément, on considère le quotient X/H d'un espace métrique géodésique propre hyperbolique X par un groupe quasi-convexe-cocompact H d'isométries de X. On montre que ce quotient est un espace hyperbolique. Le résultat principal de cette thèse indique que le nombre de bouts de l'espace quotient X/H est déterminé par les classes d'équivalence sur une sphère de rayon explicitement calculable. Dans le cadre de la théorie des groupes, on montre que l'on peut construire explicitement des groupes et des sous-groupes pour lesquels il n'existe pas d'algorithme permettant de déterminer le nombre de bouts relatifs. Si le sous-groupe est quasi-convexe, on donne un algorithme permettant de calculer le nombre de bouts relatifs. / This thesis is devoted to the study of the topology at infinity of spaces generalizing Schreier graphs. More precisely, we consider the quotient X/H of a geodesic proper hyperbolic metric space X by a quasiconvex-cocompact group H of isometries of X. We show that this quotient is a hyperbolic space. The main result of the thesis indicates that the number of ends of the quotient space X/H is determined by equivalence classes on a sphere of computable radius. In the context of group theory, we show that one can construct explicitly groups and subgroups for which there are no algorithm to determine the number of relative ends. If the subgroup is quasiconvex, we give an algorithm to compute the number of relative ends.

Type-II Ribosome Inactivating Proteins from Abrus precatorius: Cytotoxicity and Mechanism of Cell Death A/B toxins produced by bacteria and plants are among the deadliest molecules known. The plant type-II ribosome inactivating proteins (RIPs) are prototype of A/B toxins. They are two subunit proteins with a toxic A subunit that harbors an RNA N-glycosidase activity and a lectin like B subunit which allows toxin entry into cells. The toxicity of A chain is due to its RNA-N-glycosidase activity which cleaves the bond between the ribose sugar and the adenine at position 4324 as demonstrated in rat liver ribosomes. The B- chain, a lectin, binds to the cell surface receptors terminating in galactose sugars and allows toxin entry into cells. The seeds of the subtropical climber Abrus precatorius contain two RIPs: the potent toxic lectin abrin and the relatively less toxic Abrus agglutinin. The toxic property of RIPs has widespread applications in the field of agriculture and medicine. The cells of our body commit suicide in response to genetic or environmental cues by the process, apoptosis or programmed cell death which results in the safe clearance of the dead cells without affecting the extra-cellular milieu. Apoptosis is essential for development, tissue homeostasis, and defense against pathogens. It involves the interplay of multiple pathways that are initiated and executed by a family of proteases termed caspases. Several plant type-I and type-II RIPs as well as bacterial toxins have been shown to induce apoptosis in cultured cell lines. Though many agents that inhibit macromolecular synthesis in cells induce DNA fragmentation and morphological changes associated with apoptosis, the link between protein synthesis inhibition by these toxins and apoptosis remains elusive. Though extensive studies have been carried out on several RIPs for e.g. ricin and shiga toxin, only few reports are available in literature on the mechanisms of toxicity exhibited by abrin, a type-II RIP, of South-East Asian origin. Earlier studies from the laboratory have focused on the sensitivity and mechanism of abrin induced cell death in Jurkat, a cell line of haematopoietic lineage and its variants. In the same direction, the objectives of my study were: (1) To delineate the structure-function relationship of Abrus agglutinin-I in comparison with abrin, (2) To establish monoclonal antibodies to the A subunit of abrin, analyzing their neutralizing effect on abrin toxicity in vitro and in vivo and (3) To delineate the pathway and determine the kinetics of apoptosis induced by abrin on cell lines of epithelial lineage. The thesis will be presented in three four chapters. The first chapter, ‘Introduction’, begins with a brief history of RIPs, followed by the description of their distribution and classification. The transport of toxins which is a unique property of this class of proteins is discussed in detail and supported with appropriate figures. Also, information pertaining to the structure of abrin and apoptosis induced by RIPs is written in brief. In the second chapter of the thesis the structural and functional studies of Abrus agglutinin-I (APA-I) as compared to abrin are discussed. Abrin and APA-I share a high degree of homology, however, previous reports by Liu et al., indicate that APA-I is many fold less toxic in cell free systems as compared to abrin. In our studies, APA-I was found to be less toxic on cultured cell lines. The IC50 value of protein synthesis inhibition by abrin was found to be 0.4 ng/ml for both Jurkat and MCF-7 cell lines. A 20-1000 fold difference was observed in the sensitivity of these cell lines to APA-I. The extent of apoptosis induced by APA-I in A3I9.2 a caspases-8 mutant Jurkat variant cell line was comparable to abrin indicating that the apoptosis induction by APA-I might not be through the extrinsic pathway. instead, our studies showed that APA-I induced apoptosis followed the mitochondrial pathway of cell death, in a caspase dependent manner similar to that of abrin. Unlike other agglutinins like wheat germ agglutinin, the agglutinating ability of the agglutinin-I had no role in the apoptosis induced. Protein synthesis inhibition appeared to be mandatory for the apoptosis induced by APA-I. The reason for the decreased toxicity of agglutinin-I became apparent on the analysis of the crystal structure of agglutinin-I obtained by us in comparison to that of the reported structure of abrin. The substitution of Asn200 in abrin with Pro199 in agglutinin-I seems to be a major cause for the decreased toxicity. This perhaps is not a consequence of any kink formation by Pro residue in the helical segment, as reported by others earlier but due to fewer interactions that proline can possibly have with the bound substrate. Passive immuno-neutralization by administration of neutralizing antibodies is widely used as therapy against poisoning by various toxins. In case of type-II RIPs like ricin, antibodies to the toxic subunit were proven to have better protective efficacy than those to the lectin subunit. Neutralizing antibodies to abrin are not reported in literature. Therefore, a panel of monoclonal antibodies (mAbs) to the recombinant A chain of abrin was developed in our laboratory and characterized, which is presented in the third chapter of the thesis. Of these, D6F10 a high affinity antibody, exhibited neutralizing effect on abrin induced cytotoxicity on different cell lines tested. Antibodies may neutralize biological toxins in multiple ways; our studies suggested that mAb D6F10 interferes in the earliest event i.e. attachment of the toxin to the cell surface. Significantly, with the administration of mice with mAb D6F10 the prophylactic effect of the mAb could be demonstrated. In chapter 4, the sensitivity, kinetics of proteins synthesis inhibition and the mechanism of abrin induced cell death in cell lines of epithelial lineage is presented. Both sensitivity and kinetics of MCF-7/pv, Ovcar3, and T47D cells appeared comparable while, a variant culture of MCF-7 over-expressing caspases-3 was 50 times more sensitive to abrin. There was no significant difference in the binding of abrin between MCF-7/pv and MCF-7/C3+ cells. Previous studies in our laboratory indicated that abrin induced apoptosis is a caspases-3 dependent process. Also, in several systems it has been shown that caspases-3 is an indispensable molecule for apoptotic cell death. To test the absolute requirement of caspase-3, we examined abrin-induced apoptosis in a human breast cancer cell line MCF-7/pv reportedly deficient in caspases-3. Unlike other molecules like cisplatin, apoptosis induced by abrin in the MCF- 7/pv cells was found to be caspase -3 independent. However faster kinetics of apoptosis is observed, indicating that there is amplification of the apoptotic signals in the presence of caspases-3 resulting in an early onset of DNA fragmentation. The kinetics of protein synthesis inhibition and apoptosis follows similar kinetics in Jurkat cells while there is a time lapse between the two events in epithelial cells. Even with very high concentrations of abrin no detectable apoptosis was observed within 24 h in epithelial cells. The onset of fragmentation occurs after 24 h in the cell lines tested as opposed to Jurkat where it is observed as early as 6 h. Inhibition of caspases rescued the toxins from DNA fragmentation suggesting that the toxin does not cause direct nuclear damage in the cell line which does not involve the activation of caspases.

Les matériaux thermodurcissables sont naturellement des isolants électriques, limitant leurs applications dans certains domaines comme l'électronique ou l'aéronautique. Ce travail de thèse consiste à développer un nouveau matériau composite thermodurcissable en présence de charges inorganiques conductrices pour apporter des propriétés électriques sans pour autant changer notablement la viscosité du système avant la polymérisation afin de permettre l'utilisation des technologies d'infusion de résine de l'aéronautique. La stratégie des travaux est basée sur la génération d'une séparation de phase au sein du matériau et la localisation des charges conductrices aux interfaces.Cette étude est scindée deux objectifs principaux. Le premier objectif consiste à étudier un système TP/TD (thermoplastique/thermodurcissable) afin d'obtenir et contrôler une morphologie interpénétrée, selon le processus de séparation de phase induite par la polymérisation. Le second objectif consiste à étudier la localisation des charges conductrices dans un système TP/TD. Deux procédés de mise en œuvre ont été développés. Le premier procédé dit " one shot " permet de localiser les charges préférentiellement et de manière homogène dans la phase thermodurcissable et apporte une conductivité uniquement à forte concentration en particules conductrices.Une seconde méthodologie a été élaborée permettant d'obtenir un matériau biphasique dans lequel les charges sont localisées préférentiellement à l'interface du système Epoxy/thermoplastique. Des conductivités, à faible taux de charges (5 % massique), de l'ordre 10-1 S/m ont pu être atteintes avec cette méthodologie.

Les matériaux thermodurcissables sont naturellement des isolants électriques, limitant leurs applications dans certains domaines comme l’électronique ou l'aéronautique. Ce travail de thèse consiste à développer un nouveau matériau composite thermodurcissable en présence de charges inorganiques conductrices pour apporter des propriétés électriques sans pour autant changer notablement la viscosité du système avant la polymérisation afin de permettre l'utilisation des technologies d'infusion de résine de l'aéronautique. La stratégie des travaux est basée sur la génération d'une séparation de phase au sein du matériau et la localisation des charges conductrices aux interfaces.Cette étude est scindée deux objectifs principaux. Le premier objectif consiste à étudier un système TP/TD (thermoplastique/thermodurcissable) afin d’obtenir et contrôler une morphologie interpénétrée, selon le processus de séparation de phase induite par la polymérisation. Le second objectif consiste à étudier la localisation des charges conductrices dans un système TP/TD. Deux procédés de mise en œuvre ont été développés. Le premier procédé dit « one shot » permet de localiser les charges préférentiellement et de manière homogène dans la phase thermodurcissable et apporte une conductivité uniquement à forte concentration en particules conductrices.Une seconde méthodologie a été élaborée permettant d’obtenir un matériau biphasique dans lequel les charges sont localisées préférentiellement à l’interface du système Epoxy/thermoplastique. Des conductivités, à faible taux de charges (5 % massique), de l’ordre 10-1 S/m ont pu être atteintes avec cette méthodologie. / Thermosetting materials suffer from a lack of electrical conductivity. In order to overcome this barrier, a natural strategy is to introduce conductive fillers above the percolation threshold. However, addition of fillers usually leads to an increase of viscosity of the formulation which precludes infusing the resin through the porous bed of carbon fibers. In order to solve this problem, we aim at creating a two phase material and locate the fillers at the interface in order to decrease the percolation at very low values.With this view, this study is divided into two parts. The first one concerns the control of multiphase composites in order to get a co-continuous morphology by a strategy called Reaction Induced Phase Separation (RIPS). Phase diagram and influence of parameters have been studied.The second part is the formulation of a composite material (thermoplastic/thermoset) in presence of inorganic fillers. We developed two differents processes which allowed us to control fillers localisation in the blend. A process called “one shot” allows to locate homogeneously inorganic particles in epoxy phase. A second process called “premix” would preferentially locate conductive fillers at the interface of the interpenetrating system.Diffusion of particles at the interface was observed in situ during the curing of a biphasic thermoset material permitting to open the road of conducting materials and a conductivity around 10-1 S/m has been reached using as low as 5 wt% carbon black. The concept of localization of filler has been valided on several systems.

No description available.

No description available.

Serotonin ist ein Neurotransmitter im zentralen Nervensystem (ZNS), die eine Vielzahl von Funktionen in der menschlichen Physiologie hat. Serotonergen Neuronen in der Raphe-Kerne des Gehirns Unser Ziel war die Leitung der Differenzierung von embryonalen Stammzellen (ES-Zellen) eingeengt und pluripotenten Stammzellen (iPS) Zellen in eine angereicherte Population von Serotonin-produzierenden Zellen, neuartige Gene, die wesentlich für die Entwicklung zu identifizieren und die Funktion des serotonergen Systems. Zu diesem Zweck haben wir differenzierten ES-Zellen in Serotonin-produzierenden Neuronen. Verwendung von RNA zu verschiedenen Zeitpunkten im Verlauf der ES-Zelldifferenzierung wir Gene spezifisch in serotonergen Linie von Affymetrix Genarray angereichert identifiziert isoliert. Um Kandidatengene bewerten wir neu programmiert Maus und Ratte embryonale Fibroblasten zu iPS-Zellen und anschließend differenziert sie serotonergen Neuronen. Wir haben uns für Cacna2d1, für eine alpha2 / delta-Untereinheit von spannungsabhängigen Calciumkanäle als prominentesten Kandidaten unter diesen Genen kodiert. Zur Analyse der Rolle des Proteins Cacna2d1 wir verwendet Cacna2d1 Knockout-Mäusen und Morpholino-Knockdown im Zebrafisch. Wir versäumt, direkte Beteiligung der Cacna2d1 mit serotonergen Systems sehen. Allerdings Immunfärbung für Cacna2d1 in Zebrafisch zeigte zeitabhängige Muster während der frühen Entwicklung. Cacna2d1 Expression wurde in seitlichen Mittellinie Stamm gesehen; vermutlich in Neuromasten Zellen. Übereinstimmend mit ihrer Charakterisierung als Neuromasten werden diese Cacna2d1-positiven Zellen in Richtung der Schwanz der Migration. Darüber hinaus zeigte Zebrafisch gestörten Migrationsverhalten der Neuromasten nach Morpholino-Knockdown von Cacna2d1. So ist diese Studie stellte klar, dass Cacna2d1 ist für Zebrafisch Seitenlinie Entwicklung aber keinen Einfluss auf die Einrichtung des serotonergen Systems. / Serotonin is a neurotransmitter in the central nervous system (CNS), which has a wide range of functions in human physiology. Serotonergic neurons are concentrated in the raphe nuclei of the brain We aimed at directing the differentiation of embryonic stem (ES) cells and induced pluripotent stem (iPS) cells into an enriched population of serotonin producing cells to identify novel genes that are essential for the development and function of serotonergic system. To this purpose we differentiated ES cells into serotonin producing neurons. Using RNA isolated at different time points during the course of ES cell differentiation we identified genes specifically enriched in the serotonergic lineage by Affymetrix gene array. To evaluate candidate genes we reprogrammed mouse and rat embryonic fibroblast to iPS cells and subsequently differentiated them to serotonergic neurons. We selected Cacna2d1, coding for an alpha2/delta subunit of voltage dependent calcium channels as a most prominent candidate among these genes. To analyse the role of the Cacna2d1 protein we used Cacna2d1 knockout mice and morpholino-knockdown in zebrafish. We failed to see direct involvement of Cacna2d1 with serotonergic system. However immunostaining for Cacna2d1 in zebrafish revealed time-dependent pattern during early development. Cacna2d1 expression was seen in lateral midline trunk; presumably in neuromast cells. Concordantly with their characterization as neuromasts, these Cacna2d1-positive cells are migrating towards the tail. Moreover, zebrafish showed disturbed migration behaviours of neuromasts after morpholino-knockdown of Cacna2d1. Thus, this study clarified that Cacna2d1 is essential for zebrafish lateral line development but does not affect the establishment of the serotonergic system.

Proteins with selective toxicity have been investigated for use in many ways. One class of proteins, ribosome-inactivating proteins (RIPs), is found throughout the plant kingdom as well as in lower organisms like certain fungi and bacteria. These are a group of proteins that has the property of damaging the ribosomes in an irreversible manner. They are N-glycosidases that modify the 28S rRNAs to render them incapable of sustaining further translation. RIPs have been divided into two groups, i.e. type I RIPs, which are single polypeptide chains and type II RIPs, which are heterodimeric. Abrin is a type II RIP, isolated from the seeds of Abrus precatorius plant commonly known as jequirity plant. It is a heterodimeric glycoprotein consisting of an A and a B subunit linked together by a single disulfide bond. The toxicity of the protein comes from the A subunit harboring the RNA-N- glycosidase activity which catalyses the depurination of a specific adenine residue at position 4324 on the 28S rRNA. The depurination of the adenine prevents the formation of a critical stem loop structure to which the elongation factor -2 (EF-2) binds during the translocation step of the translation, thus stalling the translation machinery of the cells. The B subunit of abrin is a galactose specific lectin. The lectin activity enables the protein toxin to bind to the cell surface glycoproteins and/or glycolipids. Binding of abrin is followed by internalization of the protein by receptor mediated endocytosis and transport to the Endoplasmic reticulum (ER) by the retrograde transport pathway. Inside the ER, the single disulfide bond linking the two subunits, is reduced which is important for the A subunit toxicity. The A subunit then translocates into the cytosol using the ER-associated degradation (ERAD) pathway and cleaves the specific adenine residue on the 28S rRNA of the 60 S ribosome involved in active translation and thereby inhibiting the protein synthesis. In addition to its ability to inhibit translation, abrin induces apoptosis in cells. Earlier work from our laboratory has shown that abrin-induced apoptosis follows the intrinsic pathway of apoptotic cell death. The treated cells show mitochondrial membrane potential loss followed by caspases -9 and -3 activation and DNA fragmentation. RIPs have been used primarily in immunotherapy because of their toxicity at very low concentrations (picomolar). With the development of monoclonal antibodies as tool for targeting cell surface markers, the possibility to couple antibodies to RIPs and thus deliver the toxic protein directly to specific cells becomes feasible. Abrin, as one such potent RIP, has gained interest in the field of medicine and immunotherapeutics. Abrin can also be a candidate for use in bioterrorism and warfare. Therefore, it is very important to first understand the inhibitory effect of abrin and the extent of its toxicity on cells. Earlier studies from our laboratory have focused on the sensitivity and mechanism of cell death induced by abrin in Jurkat cells, a T –cell line. In the present study, we attempted to investigate the overall toxicity of the molecule with respect to both properties, inhibition of protein translation and induction of apoptosis, in different lineages of cells. We have carried out a comparative study on abrin toxicity on human cell lines from two different cell lineages namely hematopoietic and epithelial. The thesis is divided into introduction and two chapters. In the introduction, we have presented the general properties of this family of proteins, with a brief history; classification and distribution of plant RIPs and their enzymatic properties. The chapter also deals with possible usage of these proteins, mainly in the field of immunotherapy. We have introduced, abrin, the protein of our interest in this chapter. The structure of abrin is described and also the biological effects of the toxin are discussed in brief. The chapter one deals with the translation inhibitory property of the protein, abrin. As mentioned earlier, abrin inhibits protein synthesis via the RNA-N-glycosidase activity residing in its A-chain. We have presented the general cytotoxic pathway of type II RIPs in this chapter. It deals with the internalization and transport of the toxin to their site of action, the cytosol. As reported earlier, our results confirmed that abrin inhibited protein synthesis in all cells. Abrin mediated inhibition of translation was dose dependent. Though the inhibition was common to all the cells from both the lineages, the sensitivity of the cells towards the toxin and kinetics of this inhibition event differed significantly. The kinetics of inhibition of protein synthesis is faster in case of hematopoietic cells as compared to the epithelial cells even at lower doses of the toxin. These differences were not due to variations in the ability of protein synthesis of cells. The chapter also discusses binding of the protein to cells. Our data suggest that binding of abrin to the cells is not responsible for the variations observed in the translation inhibitory property of the protein except in Raji cells. The B-cell line Raji was found to be least sensitive towards the toxin. Our studies show that due to presence of high sialic acid residues on the surface of these cells, Raji cells are refractory to abrin mediated inhibition of protein synthesis. The second chapter presents our data on cell death upon abrin treatment. This part is divided into an introduction and two sections, A and B. In the introduction, different cell death modalities are discussed along with recent findings in the field of programmed cell death. Section A deals with abrin induced apoptosis in epithelial cells. We have compared the extent of abrin-triggered apoptosis in these cells. Some of the early events known in the apoptotic cascade of abrin are compared. Though apoptosis is observed in these cells, our data suggest a delay in the apoptotic trigger in the epithelial cells showing that epithelial cells can survive the stress induced by abrin for a longer time. When treated with other apoptotic agents, like etoposide, these cells are found to be resistant. Therefore, though there is a delay in the trigger of apoptosis, we have shown that the cells tested from the epithelial lineage undergo apoptosis on abrin treatment. Section B, discusses the ability of the protein to induce cell death in hematopoietic cells. We have presented studies on cell death other than apoptosis, detected in these cells upon abrin treatment. We found that some of the cell lines tested undergoes more necrosis than apoptosis with abrin treatment. When the status of the mitochondria was checked, we found that in U266B1 cells, a B-cell line, there was mitochondrial stress as well as reactive oxygen species (ROS) production. But these cells died by necrosis. The data obtained from this study show the involvement of lysosomes and cathepsins in abrin induced cell death in U266B1 cells. Though other cells also undergo necrosis, these events were unique to U266B1 cells.

Ribosome Inactivating Proteins (RIPs) are protein or glycoprotein toxins that bring about the arrest of protein synthesis by directly interacting with and inactivating the ribosomes. Such toxins are in general, of plant origin and differ from bacterial toxins that inhibit protein synthesis by mechanisms other than ribosome inactivation. After the toxins had been in the centre of interest in biomedical research for a couple of decades in the end of 19th century, the scientific community largely lost interest in the plant toxins. Interest in these toxins was revived when it was found that they are more toxic to tumor cells when compared to normal cells. Based on their structure RIPs can be classified into three types: Type I RIPs – They consist exclusively of a single RNA-N-glycosidase chain of ~30kDa. Type II RIPs – They consists of chain-A comparable to type I RIPs linked by a disulfide bridge to an unrelated chain-B, which has carbohydrate binding activity. The molecular weight of the type II RIPs is ~60kDa. Type III RIPs – Besides the classical type II RIPs a 60kDa RIP (called JIP60) has been identified in barley (Hordeum vulgare) that consists of chain-A resembling type I RIPs linked to an unrelated chain-B with unknown function. In addition to these classes of RIPs there is another group of toxins called four subunit toxins, whose structure is almost similar to type II RIPs, but are made up of two such subunits linked by non-covalent interactions forming tetramers having two A- and two B-chains. The definition and classification of these toxins is not so clear as they are frequently referred to as agglutinins or lectins (e.g Abrus precatorius agglutinins I and II, Ricinus communis agglutinin etc.), having red blood cell (RBC) agglutinating activity. However they have been found to be less toxic and better agglutinins when compared with type II RIPs. The present thesis reports the crystal structure of a type II RIP, Abrus precatorius agglutinin-I (APA-I) from the seeds of Abrus precatorius plant. The protein was purified from the plant seed and crystallized. The crystal structure was solved by molecular replacement method. Preliminary crystals of abrus agglutinin were obtained almost thirty years ago and unsuccessful attempts to solve the crystal Structure of APA-I were made almost five years ago by other groups. The structure solution of API-I was obtained at 3.5 Å using synchrotron data set collected at room temperature from a single crystal. Crystal structure is already known for Abrin, another type II RIP isolated from the same seeds. Abrin and APA-I have similar therapeutic indices for the treatment of experimental mice with tumors, but APA-I has much lower toxicity, with lethal dose (LD50) being 5mg/kg of body weight when compared with Abrin-a (LD50 = 20 μg/kg of body weight). The striking difference in the toxicity shown by Abrin and its agglutinin (APA-I) encouraged us to look at the structure function relationship of these proteins, which might prove to be useful in the design and construction of immunotoxins. As apparent from the comparative study, the reduced toxicity of APA-I can be attributed to fewer interactions it can possibly have with the substrate due to the presence of Pro199 at the binding site and not due to any kink formed in the helix due to the presence of praline as reported by other groups. In recent years, these plant RIPs which inhibit protein synthesis have become a subject of intense investigation not only because of the possible role played by them in synthesizing immunotoxins that are used in cancer therapy but also because they serve as model system for studying the molecular mechanism of transmembrane translocation of proteins. In silico docking studies were carried out in search of inhibitors that could modulate the toxicity of RIPs. Many adenine like ringed compounds were studied in order to identify them as novel inhibitors of Abrin-a molecule and facilitate detailed analyis of protein ligand complex in various ways to ascertain their potential as ligands. In addition, the structural analysis of conformationally constrained, α β-dehydrophenylalanine containing dipeptides is carried out. While there are several studies of molecular self assembly of peptides containing coded amino acids, not much work has been done on molecular assembly formation utilizing non-coded amino acids. The non-coded amino acid used in the analysis is a member of α β-dehydroamino acids. These are the derivatives of protein amino acids with a double bond between Cα And Cβ atoms and are represented by a prefix symbol ‘Δ’. They are frequently found in natural peptides of microbial and fungal origins. The presence of α , β-dehydroamino acid residues in bioactive peptides confers altered bioactivity as well as an increased resistance to enzymatic degradation. Thus, α, β-dehydroamino acid residues, in particular α, β-dehydrophenylalaine(ΔPhe) has become one of the most promising residues in the study of structure-activity relationships of biologically important peptides. The utilization of in the molecular self assembly ΔPhe in the molecular self assembly offers in added benfit in terms of variey and stability. Taking advantage of the conformation constraining property of the ΔPhe residue, its incorporation in three dipeptide molecules has been probed. In this thesis the crystal structures of the following designed dipeptide are reported.(I). +H3N-Phe-ΔPhe-COO˙ (FΔF); (II). +H3N-Val-ΔPhe- COO˙ (VΔF); +H3N-Ala-ΔPhe-COO˙ (AΔF). The peptides were found to be in the zwitterionic conformation and two (I, II) of the three dipeptides have resulted in tubular structures of dimensions in the nanoscale range. Chapter 1 starts with a brief introduction of RIPs, their classification and overall fold, with Abrin-a as example. A brief mention is made about how the protein is translocated in the cell and the depurination mechanism. Chapter 2 presents the purification of APA-I from the seeds of Abrus precatorius plant, the crystallization of APA-I, X-ray intensity data collection on these crystals and processing of data sets for APA-I. Chapter 3 details the structure determination of tetramer Abrus precatorius agglutinin-I,(APA-I), using the molecular replacement method, iterative model building and refinement and the quality of final protein structure model. Chapter 4 details the crystal structure of Abrus precatorius agglutinin-I (APA-I), the comparison of primary and secondary structure of APA-I with Abrin-a and the structural insights into the reduced toxicity in relation to Abrin-a and future prospects. Chapter 5 deals with the in-silico modeling of Abrin-a inhibitors using the docking method. Abrin-a is being tested extensively for the design of therapeutic immunotoxins. Chapter 6 deals with the self-assembly of dipeptides containing conformationally constrained amino acid, α. β -dehydrophenylalanine (ΔF).

Type II Ribosome Inactivating Proteins (RIPs), commonly known as A/B toxins are heterodimers comprising of a catalytically active A chain, an RNA N-glycosidase which inhibits protein synthesis and a lectin-like B chain required for the binding of the toxin to the cell surface and internalization of the same. Abrin is a type II RIP obtained from the mature seeds of Abrus precatorius plant that is extremely toxic and has been shown to be 75 times more potent than its well studied sister toxin, ricin. The LD50 dose for abrin is only 2.8 µg/kg body weight of mice and its potential use in bio-warfare is a cause of major concern. Abrin has been classified as a select agent by the Centre for Disease Control and Prevention, U.S.A., because it is stable, effective at very low concentrations and easy to purify and disseminate in large amounts. In spite of abrin being a potential bio-warfare agent, there is no antidote or vaccine available against this toxin till date. The first and only neutralizing monoclonal antibody (mAb) against abrin, namely D6F10, was reported from our laboratory and has been shown to rescue toxicity of abrin in cells as well as in mice. The study reported in the thesis focuses on understanding the mechanism of neutralization of abrin by the mAb D6F10 and development of a potential vaccine candidate against the toxin. In order to map the epitope corresponding to the antibody, first, overlapping gene deletion constructs spanning the entire length, 251 amino acids, of ABA were generated and checked for binding to the mAb. Fragments shorter than 1-175 did not show immuoreactivity. Analysis of the crystal structure of abrin A chain revealed that a helix spanning the amino acids 148-167 was present at the core of the protein structure and truncation in this region of the protein possibly results in loss of conformation leading to abrogation of antibody binding. Therefore, a novel strategy of epitope mapping was adopted. Abrus precatorius agglutinin (APA) is a homologue of abrin obtained from the same plant source. The A chains of abrin and APA share 67% sequence identity and their crystal structures superimpose very well but unlike abrin the APA A chain does not bind the mAb D6F10. Chimeric constructs were generated within the region 1-175 of A chains of both ABA and APA and deletions and mutations of the ABA was then made on the APA as scaffold. It could be concluded that the amino acids of the region 75¬123 are involved in the formation of the epitope. Further, based on sequence alignment of ABA and APA A chain 13 residues in the chimera ABA1-123APA124-175 were mutated and it was found that the mutation of the residues Thr 112, Gly 114 and Arg 118 resulted in loss of binding to the antibody. Furthermore, the mAb D6F10 rescues inhibition of protein synthesis by abrin in HeLa cells by internalizing in cells along with abrin and possibly occluding the active site cleft of ABA. The antibody prevents cell attachment of abrin at higher concentrations. The observations provide novel insights into mechanisms of many known neutralizing antibodies against A/B toxins. The study also highlights that chimeric protein constructs could possibly be developed as potential vaccine candidates for neutralization of abrin intoxication.