Lectins are multivalent carbohydrate binding proteins that specifically recognise diverse sugar structures and mediate a variety of biological processes, such as cell-cell and host-pathogen interactions, serum glycoprotein turnover and innate immune responses. Lectins have received considerable attention in recent years on account of their properties which have led to their wide use in research and biomedical applications. Seeds of leguminous plants are rich sources of lectins, but they are also found in all classes and families of organisms. Legume lectins have similar tertiary structures, but exhibit a large variety of quaternary structures. The carbohydrate binding site in them is made up of four loops, the first three of which are highly conserved in all legume lectins. The fourth loop, which is variable, is implicated in conferring specificity. Legume lectins which share the same monosaccharide specificity often exhibit markedly different oligosaccharide specificities. The introductory chapter gives a broad overview of lectins from a structural point of view. The rest of the thesis is primarily concerned with structural studies on lectins from seeds of the winged bean (Psophocarpus tetragonolobus). Winged bean seeds contain a basic lectin (WBAI) (pi > 9.5) and an acidic lectin (WBAII) (pi -5.5). Both these lectins are N-glycosylated homodimers with about 240 amino acid residues per monomer. They show a high affinity for methyl-a-D-galactose at the monosaccharide level but have entirely different affinities for oligosaccharides. WBAI agglutinates human type A and B erythrocytes but not O type, while WBAII binds specifically to the terminally monofucosylated H-antigenic (responsible for O blood group reactivity) determinants on the cell surface. In this context, the current study seeks to characterise the carbohydrate binding site of a saccharide-free form of WBAI and determine the structural basis of carbohydrate recognition in WBAII. The study also aims to identify the factors responsible for the differences in carbohydrate specificities between WBAI and WBAII. Diffraction data from a saccharide-free crystal form of WBAI and two crystal forms (Form I and II) of WBAII complexed with methyl-a-D-galactose were collected on a MAR imaging plate system mounted on a Rigaku RU200 rotating anode X-ray generator. The data were processed using the MAR-XDS and DENZO/SCALEPACK suites of programs. The structures were solved by the molecular replacement method using AMoRe. The model used in the case of WBAI and Form I of WBAII was the structure of WBAI in complex with methyl-a-D-galactose (PDB coderlWBL), while the structure of Form II of WBAH was solved using a partially refined model of Form I. The refinements and model building were performed using the programs X-PLOR/CNS and O respectively. A comparison of the structures of the saccharide-free and bound forms of WBAI revealed three water molecules occupying the carbohydrate binding site, which mimic the hydrogen bonded interactions made by the saccharide in the structure of the complex. Also a shift of -0.6 A in the variable loop, towards the saccharide in the structure of the complex was observed. Significant differences in the conformation of a loop involved in crystal packing interactions were also observed. An analysis of protein hydration demonstrates, among other things, the role of water molecules in stabilising the structure of the loops around the carbohydrate binding site. The crystal structures of the two forms of WBAH were solved at 3.0 A and 3.3. A resolution. The structure of the complex revealed the role of the length of the variable loop in generating the difference in oligosaccharide specificity between WBAI and WB All. The difference in the pi values between the two lectins is caused by substitutions occurring in loops and edges of sheets. A distinct structural difference between WBAH and all the other legume lectins of known structure is in the new disposition of the 34-45 loop with an r.m.s deviation of -6.0A in Coc positions compared to its position in other lectins. This change in conformation is caused by the formation of salt bridges by amino acid residues unique to WB All in the 34-45 loop and its neighbourhood. Thermodynamic studies on the binding of H-antigenic determinant to WBAII showed a predominance of entropic contribution suggesting a hydrophobically driven binding, not yet observed in lectin-sugar interactions. An analysis involving the docking of H-type II trisaccharide (Fuca(l-2)Galf}(l-4)GlcNAc) into the carbohydrate binding site and a comparison with the binding sites of other legume lectins revealed the role of a Tyr in the variable loop and an Asn in the second loop that are unique to WBAII in generating this unique binding property. Earlier work on peanut lectin and WBAI demonstrated that the modes of dimerisation of legume lectins are governed by features intrinsic to the protein. A phylogenetic analysis of the sequences of all legume lectins whose structures are available has been performed to examine the relationship among the various classes of oligomers and classes of sugar specificity. The information thus obtained showed that groups of legume lectins that share a common mode of dimerisation cluster together. A sequence alignment based on structures revealed amino acid residues unique to each of these clusters that may be important in determining the modes of observed dimerisation. While pursuing structural studies on WBAI and WBAII, the author has also been involved in an ongoing small molecule project in the laboratory, which involves preparation and X-ray structure determination of the complexes of carboxylic acids with amino acids and peptides. The work carried out in the project is described in the appendix.

I formally joined Prof. M. R. N. Murthy’s laboratory at the Molecular Biophysics Unit, Indian institute of Science, on 1st August 2001. During that time, the interest in the laboratory was mainly focused on structural studies on a number of capsid mutants of two plant viruses, sesbania mosaic virus and physalis mottle virus, to gain an insight into the virus structure and its assembly. Besides these two projects, there were a few other collaborative projects running in the lab at that time such as NIa protease from pepper vein banding virus and diaminopropionate ammonia lyase from Escherichia coli with Prof. H. S. Savithri, triosephosphate isomerase from Plasmodium falciparum with Prof. P. Balaram and Prof. H. Balaram and a DNA binding protein (TP2) with Prof. M. R. S. Rao. During my first semester, along with my course work, I was assigned to make an attempt to purify and crystallize recombinant NIa protease and TP2 protein. I started with NIa protease which could be purified using one step Ni-NTA affinity column chromatography. Although the expression and protein yield were reasonably good, protein precipitated with in a couple of hours after purification. Attempts were made to prevent the precipitation of the purified enzyme and towards this end we were successful to some extent. However, during crystallization trials most of the crystallization drops precipitated completely even at low protein oncentration. TP2 protein was purified using three-step chromatographic techniques by one of the project assistant in Prof. M. R. S. Rao’s laboratory. Because of low expression level and three step purification protocol, protein yield was not good enough for complete crystallization screening. Hits obtained from our initial screening could not be confirmed because of low protein yield as well as batch to batch variation. My attempts to crystallize these two proteins remained unsuccessful but in due course I had learnt a great deal about the tips and tricks of expression, purification and mainly crystallization. To overcome the problems faced with these two proteins, we decided to make some changes in the gene construct and try different expression systems. By this time (beginning of 2002), I had finished my first semester and a major part of the course work, so we decided to start a new project focusing on some of the unknown enzymes from a metabolic pathway. Dr. Parthasarathy, who had finished his Ph. D. from the lab, helped me in literature work and in finding targets for structural studies. Finally, we decided to target enzymes involved in the propionate etabolism. The pathways for propionate metabolism in Escherichia coli as well as Salmonella typhimurium were just established and there were no structural information available for most of the enzymes involved in these pathways. Since, propionate metabolic pathways were well described in the case of Salmonella typhimurium, we decided to use this as the model organism. We first started with the enzymes present in the propionate catabolic pathway “2-methylcitrate pathway”, which converts propionate into pyruvate and succinate. 2-methylcitrate pathway resembles the well-studied glyoxylate and TCA cycle. Most of the enzymes involved in 2-methylcitrate pathway were not characterized biochemically as well as structurally. First, we cloned all the four enzymes PrpB, PrpC, PrpD and PrpE present in the prpBCDE operon along with PrpR, a transcription factor, with the help of Dr. P.S. Satheshkumar from Prof. H. S. Savithri’s laboratory. Since these five proteins were cloned with either N- or C-terminal hexa-histidine tag, they could be purified easily using one-step Ni-NTA affinity column chromatography. PrpB, PrpC and PrpD had good expression levels but with PrpE and PrpR, more than 50% of the expressed protein went into insoluble fraction, probably due to the presence of membrane spanning domains in these two enzymes. Around this time, crystallization report for the PrpD from Salmonella was published by Ivan Rayment’s group, so after that we focused only on the remaining four proteins leaving out PrpD. Our initial attempts to crystallize these proteins became successful in case of PrpB, 2-methylisocitrate lyase. We collected a complete diffraction data to a resolution of 2.5 Å which was later on extended to a resolution of 2.1 Å using another crystal. Repeated crystallization trials with PrpC also gave small protein crystals but they were not easy to reproduce and size and diffraction quality always remained a problem. Using one good crystal obtained for PrpC, data to a resolution of 3.5 Å could be collected. Unfortunately, during data collection due to failure of the cryo-system, a complete dataset could not be collected. Further attempts to crystallize this protein made by Nandashree, one of my colleagues in the lab at that time, was also without much success. Attempts to purify and crystallize PrpE and PrpR were made by me as well as one of my colleagues, Anupama. In this case, besides crystallization, low expression and precipitation of the protein after purification were major problems. Our attempt to phase the PrpB data using the closest search model (phosphoenolpyruvate mutase) by molecular replacement technique was unsuccessful,probably because of low sequence identity between them (24%). Further attempts were made to obtain heavy atom derivatives of PrpB crystal. We could obtain a mercury derivative using PCMBS. However, an electron density map based on this single derivative was not nterpretable. Around this time, the structure of 2-methylisocitrate lyase (PrpB) from E. coli was published by Grimm et. al. The structure of Salmonella PrpB could easily be determined using the E. coli PrpB enzyme as the starting model. We also solved the structure of PrpB in complex with pyruvate and Mg2+. Our attempts to crystallize PrpB with other ligands were not successful. Using the structures of PrpB and its complex with pyruvate and Mg2+, we carried out comparative studies with the well-studied structural and functional homologue, isocitrate lyase. These studies provided the plausible rationale for different substrate specificities of these two enzymes. Due to unavailability of PrpB substrate commercially and the extensive biochemical and mutational studies carried out by two different groups made us turn our attention to other enzymes in this metabolic pathway. Since our repeated attempts to obtain good diffraction quality crystals of PrpC, PrpE and PrpR continued to be unsuccessful, we decided to target other enzymes involved in propionate metabolism. We looked into the literature for the metabolic pathways by which propionate is synthesized in the Salmonella typhimurium and finally decided to target enzymes present in the metabolic pathway which converts L-threonine to propionate. Formation of propionate from L-threonine is the most direct route in many organisms. During February 2003, we initiated these studies with the last enzyme of this pathway, propionate kinase (TdcD), and within a couple of months we could obtain a well-diffracting crystal in complex with ADP and with a non-hydrolysable ATP analog, AMPPNP. TdcD structure was solved by molecular replacement using acetate kinase as a search model. Propionate kinase, like acetate kinase, contains a fold with the topology βββαβαβα, identical with that of glycerol kinase, hexokinase, heat shock cognate 70 (Hsc70) and actin, the superfamily of phosphotransferases. Examination of the active site pocket in propionate kinase revealed a plausible structural rationale for the greater specificity of the enzyme towards propionate than acetate. One of the datasets of TdcD obtained in the presence of ATP showed extra continuous density beyond the γ-phosphate. Careful examination of this extra electron density finally allowed us to build diadenosine tetraphosphate (Ap4A) into the active site pocket, which fitted the density very well. Since the data was collected at a synchrotron source to a resolution of 1.98 Å, we could identify the ligand in the active site pocket solely on the basis of difference Fourier map. Later on, co-crystallization trials of TdcD with commercially available Ap4A confirmed its binding to the enzyme. These studies suggested the presence of a novel Ap4A synthetic activity in TdcD, which is further being examined by biochemical experiments using mass-spectrometry as well as thin-layer chromatography experiments. By the end of 2004, we shifted our focus to the first enzyme involved in the anaerobic degradation of L-threonine to propionate, a biodegradative threonine deaminase (TdcB). Sagar Chittori, who had joined the lab as an integrated Ph. D student, helped me in cloning this enzyme. My attempt to crystallize this protein became finally successful and datasets in three different crystal forms were collected. Dataset for TdcB in complex with CMP was collected during a synchrotron trip to SPring8, Japan by my colleague P. Gayathri and Prof. Murthy. TdcB structure was solved by molecular replacement using the N-terminal domain of biosynthetic threonine deaminase as a search model. Structure of TdcB in the native form and in complex with CMP helped us to understand several unanswered questions related to ligand mediated oligomerization and enzyme activation observed in this enzyme. The structural studies carried out on these three enzymes have provided structural as well as functional insights into the catalytic process and revealed many unique features of these metabolic enzymes. All these have been possible mainly due to proper guidance and encouragement from Prof. Murthy and Prof. Savithri. Prof. Murthy’s teaching as well as discussions during the course of investigation has helped me in a great deal to learn and understand crystallography. Collaboration with Prof. Savithri kept me close to biochemistry and molecular biology, the background with which I entered the world of structural biology. The freedom to choose the project and carry forward some of my own ideas has given me enough confidence to enjoy doing research in future.

François Couperin (1668-1733) was one of the earliest French Baroque composers to merge the Italian style into the French tradition. He had great influence on the development of French Baroque music from the end of the seventeenth century until his death. Couperin's four Concerts Royaux and the ten Concerts Nouveaux (published in 1722 and 1724) were written for the enjoyment of Louis XIV. Those suites were popular in the court before they were published, as they were requested to be performed every Sunday during the years 1714 and 1715 to give pleasure to the king. Rittrato dell'amore is the ninth suite out of the fourteen suites. The purpose of this study is to provide a performance guide and a practical edition of François Couperin's Neuvième Concert Ritratto dell' amore. It also contrasts Italian style and French tradition in the Baroque period, and how Couperin blended both styles together in his Neuvième Concert. In addition, this dissertation summarizes the general principles of Baroque performance practice that one may encounter in Neuviéme Concert, including notes inégales (unequal notes), ornamentation, over-dotting, and other issues. It is especially important for one to understand the performance style of French Baroque music in order to perform these works appropriately, since its notation did not adequately notate rhythmic expectations as traditionally understood and the realization of ornamentations in this period and style is highly specific. The tradition was indeed lost in terms of aural transfer and has been reconstructed through published scholarly work in the last century that is based on treatises of the time. Ongoing scholarly and artistic work should bring us ever closer to the ideals of the period.

Il presente lavoro ha per oggetto la filosofia morale di Vladimir Jankélévitch e, specificatamente, l’arco teorico che dalla metafisica porta all’etica. Tale lavoro mira a mettere a fuoco la domanda fondamentale della morale di Jankélévitch e, contestualmente, a mostrarne l’ancoramento nella prospettiva metafisica dell’Autore. L’obiettivo ultimo della ricerca, in sintesi, è rendere ragione della coincidenza tra morale e filosofia prima teorizzata da Vladimir Jankélévitch. L’ipotesi guida della ricerca è che il fondamento di tale coincidenza sia da rintracciare nelle pagine di Philosophie première, testo del 1954 in cui Jankélévitch compie due mosse teoriche decisive. In primo luogo, presenta l’Assoluto come Faire-être sans être e secondariamente lo qualifica come atto puro rispetto al quale l’atto umano è impuro. Unitamente alla ricostruzione della proposta metafisica dell’Autore, allora, è l’analisi del significato filosofico degli aggettivi puro e impuro che ha permesso di mostrare due punti. Innanzitutto, che la domanda di fondo di Jankélévitch in sede morale riguarda la possibilità per l’uomo di fare esperienza dell’Assoluto. In secondo luogo, che la risposta di Jankélévitch a tale interrogativo è affermativa e che l’articolazione di tale riposta passa attraverso la nozione di sacrificio per amore. / This research focuses on the moral philosophy of Vladimir Jankélévitch and, in particular, on the theoretical relation between metaphysics and morality. This work aims to clarify the essential question of Jankélévitch’s moral philosophy and, at the same time, to show the close link of this question with Jankélévitch’s metaphysical perspective. The main aim of this research is to present and to explain the reasons why Jankélévitch asserts that ethics is first philosophy. The core hypothesis is that the foundation of this assessment could be found in the pages of Philosophie première, where, in 1954, Jankélévitch proposes two decisive arguments. First of all, he presents the Absolute as Faire-être sans être and, secondly, he qualifies the Absolute as pure Act with regard to which every human act is impure. In addition to the critical presentation of Jankélévitch’s metaphysics, the analysis of the philosophical meaning of the adjectives pure and impure allows to show two points. Firstly, it shows that the fundamental question of Jankélévitch’s ethics concerns the human possibility to experience the Absolute. Secondly, it demonstrates that Jankélévitch’s answer to this question is affirmative and it is centred on the notion of loving sacrifice.

Edmund Husserl tratta l’etica e l’intersoggettività separatamente e con scopi diversi. Allo stesso tempo, nei suoi testi sono presenti indizi di una mutua connessione tra tali ambiti. Nel mio lavoro intendo chiarire come sia interpretabile tale connessione. In particolare, considero che l’affermazione husserliana del valore della relazione e del dovere di promuoverla indica che il soggetto può realizzare attivamente la dimensione intersoggettiva in cui si trova solo attraverso una scelta a favore della relazione. Tale scelta presuppone l’esperienza del valore altrui. Mi chiedo quindi quali siano le condizioni dell’esperienza della rilevanza assiologica ed etica dell’altro e della relazione con lui. Per rispondere a questa domanda, mi rivolgo a due ambiti della fenomenologia trascendentale di Husserl: la teoria dell’intersoggettività e le analisi etiche. Attraverso la prima, valuto se gli strati costitutivi dell’esperienza intersoggettiva abbiano una rilevanza assiologica. Tuttavia, dati i limiti di una considerazione etica della teoria dell’intersoggettività, mi rivolgo alle analisi etiche per ulteriori approfondimenti. Esse mostrano che il valore dell’altro e il dovere nei suoi confronti sono colti dall’atto emotivo dell’amore e che l’amore a sua volta è fondato nel coglimento dell’altro come soggetto trascendentale. Così facendo chiarifico la mutua connessione tra etica e teoria dell’intersoggettività in Husserl. / Edmund Husserl treats ethics and intersubjectivity separately and with different purposes. At the same time, he disseminates clues of their interconnectedness throughout his works. In my dissertation, I aim to elucidate how to interpret their connection. In particular, I argue that Husserl’s insistence on the value of relationship and on the duty to promote it indicates that the subject can actively realize the intersubjective dimension in which it is situated only through a choice in favour of the relationship. This choice presupposes the experience of the value of the other. I thus ask which are the conditions of the experience of the axiological and ethical relevance of the other and of the relationship with it. To answer this question, I turn to two areas of Husserl’s transcendental phenomenology: his theory of intersubjectivity and his ethical analyses. Through the first, I assess whether the constitutive levels of intersubjective experience have axiological relevance. However, given the limits pertaining to an ethical consideration of his theory of intersubjectivity, I move to Husserl’s ethical analyses for further insights. These analyses show that the value of the other and the duty towards it are captured by the emotional act of love, and that love is in turn grounded on grasping the other as a transcendental subject. I thereby shed light on the interconnectedness of Husserl’s ethics and theory of intersubjectivity.