The thesis entitled “New DNA-Targeting Small Molecules as Potential Anticancer Agents and for in vivo Specificity toward Enhanced Silk Production” encompasses design, computational calculations, and syntheses of diverse small molecular scaffolds to explicitly target duplex and higher order DNA morphologies (G-quadruplex DNA). Some of these molecules have a potential as anticancer agents. Besides, an attempt has been made elucidate the importance of novel oligopyrrole carboxamides in the enhancement of silk yield, hence proving to a boon in the field of sericulture. The work has been divided into six chapters. Chapter 1. DNA Binding Small Molecules as Anticancer Agents Figure 1. DNA targeting by small molecules. Cancer has always been a dreadful disease and continues to attract extensive research investigations. Various targets have been identified to restrain cancer. Among these DNA happens to be the most explored one. A wide variety of small molecules, often referred to as “ligands”, has been synthesized to target numerous structural features of DNA (Figure 1). The sole purpose of such molecular design has been to interfere with the transcriptional machinery in order to drive the cancer cell toward apoptosis. The mode of action of the DNA targeting ligands focuses either on the sequence-specificity by groove binding and strand cleavage, or by identifying the morphologically distinct higher order structures like that of the G-quadruplex DNA. Chapter 2. Ligand 5, 10, 15, 20-tetra(N-methyl-4-pyridyl)porphine (TMPyP4) Prefers the Parallel Propeller-type Human G-Quadruplex DNA over its other Polymorphs The binding of ligand 5, 10, 15, 20-tetra(N-methyl-4-pyridyl)porphine (TMPyP4) with telomeric and genomic G-quadruplex DNA has been extensively studied. However, a comparative study of interactions of TMPyP4 with different conformations of human telomeric G-quadruplex DNA, namely parallel propeller-type (PP), antiparallel basket-type (AB), and mixed hybrid-type (MH) G-quadruplex DNA has not been done. We considered all the possible binding sites in each of the G-quadruplex DNA structures and docked TMPyP4 to each one of them. The resultant most potent sites for binding were analyzed from the mean binding free energy of the complexes. Molecular dynamics simulations were then carried out and analysis of the binding free energy of the TMPyP4-G-quadruplex complex showed that the binding of TMPyP4 with parallel propeller-type G-quadruplex DNA is preferred over the other two G-quadruplex DNA conformations. The results obtained from the change in solvent excluded surface area (SESA) and solvent accessible surface area (SASA) also support the more pronounced binding of the ligand with the parallel propeller-type G-quadruplex DNA (Figure 2). Figure 2. Ligand TMPyP4 prefers parallel propeller-type G-quadruplex DNA morphology. Chapter 3. A Theoretical Analysis on the Selective Stabilization of Intermolecular G-quadruplex RNA with a bis-Benzimidazole Ligand EtBzEt over TMPyP4 in K+ Environment Ever since the discovery of G-quadruplex RNA, a constant urge exists to target these higher order RNA conformations. These structures play a significant role in the transcriptional and translational mechanism. Herein we have determined the mode and extent of association of certain G-quadruplex DNA binding bisbenzimidazole ligand (EtBzEt) in comparison to a known porphyrin ligand (TMPyP4). We have performed docking studies of the known G-quadruplex DNA binding ligands with the parallel propeller G-quadruplex RNA (PPR) to determine the most potent binding conformation which showed EtBzEt to be a better RNA binder than others. Furthermore, a molecular dynamics (MD) simulation (6 ns) was performed for the most stable docked complex in explicit solvent environment. The role of K+ ions, Hoogsteen hydrogen bond formation and backbone dihedral angle between the tetrads were carefully analyzed during the entire simulation run to determine the stability of each ligand associated PPR complex. All the analyses conclusively showed that while TMPyP4 merely stabilized the PPR, the ligand EtBzEt stabilized PPR very efficiently (Figure 3). Figure 3. Stabilzation and destabilization by EtBzEt and TMPyP4, repectively. Red and green ovals represent EtBzEt and TMPyP4, repectively. Chapter 4A. Design and Synthesis of New DNA Binding Fe(III) and Co(II) Salen Complexes with Pendant Oligopyrrole Carboxamides Extensive research on these oligopyrrole carboxamides has shown their specificity toward AT-rich sequences with high binding affinity. Here we have designed and synthesized Fe (III)-and Co (II)-based salen complexes attached with minor groove targeting oligopyrrole carboxamide side-chains (Figure 4). While the ligands showed excellent activity toward DNA damage, they also exhibited high affinity toward the minor grooves of the ds-DNA. This was also reflected in the high efficiency of the ligands toward cancer cell cytotoxicity. Further studies revealed that the ligands resulted in prominent nuclear condensation and fragmentation thereby driving the cells toward apoptosis. The presence of metal coordinated salen moiety conjugated with positively charged pendants ending with minor groove binding oligopyrrole carboxamides might have resulted in the increased activity of the ligands toward DNA targeting and cancer cell death. Figure 4. Chemical structures of the ligands used in this study. Chapter 4B. Design and synthesis of novel oligopyrrole based salen metal complexes and their efficiency toward stabilization of G-quadruplex DNA DNA targeting has been the key strategy toward the restriction of cancer cell proliferation. In a similar effort, we have designed and synthesized novel salen based Ni(II) and Pd(II) metal complexes with positively charged flanking side-chains comprising attached N-methylpyrrole carboxamides of varying lengths (Figure 5). The ligands showed efficient stabilization of the G-quadruplex DNA morphologies, with specificity over the duplex DNA. Sufficient inhibition of the telomerase activity was observed by the TRAP-LIG assay which was ascertained by the prominent restriction of cancer cell proliferation in the long-term cell viability assay. The ligands exhibited condensation and fragmentation of the nucleus when observed under confocal microscopy which is indicative of the cells undergoing apoptosis. Further annexin V-FITC and PI dual staining showed apoptosis to be the mechanistic pathway underlying the cancer cell cytotoxicity by the ligands. Modeling studies clearly showed the stacking of the salen moiety over the G-tetrads with the association of the pendant oligopyrrole carboxamide units to the grooves. Figure 5. Chemical structures of the ligands used in this study. Chapter 5A. Role of Metal Ions in Novel Fluorescein based Salen and Salphen Complexes toward Efficient DNA Damage and their Effect on Cancer Cells Metal ions play an important role toward DNA damage and numerous ligands have been synthesized for their use in anticancer therapy. Herein, we have designed and synthesized Fe(III) and Co(II) based salen/salphens by bridging two fluorescein moieties with varying spacers (Figure 6). Although the ligands exhibit dual binding mode, the more flexible salen ligands prefer to associate to the minor groove of the DNA while the relatively rigid salphen ligands show greater intercalation. The biophysical experiments reveal better binding affinity of the salphens toward duplex DNA as compared to the salen ligands. The metal coordination resulted in efficient DNA cleavage of plasmid at low ligand concentrations. The ligands also showed cancer cell cytotoxicity, cellular internalization with apoptosis as the proposed mechanism for cell death. Figure 6. Chemical structures of the salen and salphen ligands used in this study. Chapter 5B. Fluorescein based Salen and salphen Complexes as stabilizers of the Human G-quadruplex DNA and Promising Telomerase Inhibitors Metal based salen complexes have been considered as an important scaffold toward targeting of DNA structures. In the present work we have designed and synthesized nickel(II)-and palladium(II)-salen and salphen ligands by using fluorescein as the backbone to provide an extended aromatic surface (Figure 7). The ligands exhibit sufficient affinity toward the human telomeric G-quadruplex (G4) DNA in preference to the duplex DNA and also exhibit promising inhibition of telomerase activity. This is ascertained by their potency in the long-term cell viability assay which shows significant cancer cell cytotoxicity in presence of the ligands. Confocal microscopy showed cellular internalization followed by nuclear localization. Considerable population at the sub-G1 phase of the cell cycle showed cell death via apoptotic pathway. Figure 7. Chemical structures of the ligands used in this study. Chapter 6. Knockdown of Broad-Complex Gene Expression of Bombyx mori by Oligopyrrole carboxamides Enhances Silk Production Bombyx mori (B. mori) is important due to its major role in the silk production. Though DNA binding ligands often influence gene expression, no attempt has been made to exploit their use in sericulture. The telomeric heterochromatin of B. mori is enriched with 5′-TTAGG-3′ sequences. These sequences were also found to be present in several genes in the euchromatic regions. We examined three synthetic oligopyrrole carboxamides that target 5′-TTAGG-3′ sequences in controlling the gene expression in B. mori (Figure 8). The ligands did not show any defect or feeding difference in the larval stage, crucial for silk production. The compounds caused silencing of various isoforms of the broad-complex transcription factor and cuticle proteins which resulted in late pupal developmental defects. This study shows for the first time use of oligopyrrole carboxamide drugs in controlling gene expression in B. mori and their long term use in enhancing silk production. Figure 8. Chemical structures of the ligands used in this study (top) and increased cocoon size on ligand treatment.

The Puritan attitude toward death in seventeenth- and early eighteenth-century New England was ambivalent and contained both terror at the possibility of eternal damnation and hope for deliverance. The joyful theme of the migratio ad Dominum resonated with the Saints only at times when they were convinced divine grace was actively working in their lives, but when they saw they were backsliding, the horror of death prevailed. Puritan anxiety about death was caused by tensions inherent in the doctrine of predestination, which implied man's dependence on God's inscrutability, and in the doctrine of assurance, which implied that self-doubt was more desirable than full assurance of salvation. What complicated any verification of the presence of grace was man's endless potential for self-deception. Memento mori gave urgency to the Puritan work ethic and the effective use of time. The anxiety about one's destiny began in early childhood when death and its ensuing horrors for the depraved were used as a means of religious instruction to provoke spiritual precocity and conversion. This early immersion into the discourse about death has been erroneously interpreted as a proof of the non-existence of childhood in Puritan New England. Deathbed scenes depicted in Puritan spiritual biographies were designed as examples...

Cette thèse est consacrée à l'étude de la géométrie des variétés de Fano complexes en utilisant les propriétés des sous-faisceaux du fibré tangent et la géométrie du diviseur fondamental. Les résultats principaux compris dans ce texte sont : (i) Une généralisation de la conjecture de Hartshorne: une variété lisse projective est isomorphe à un espace projectif si et seulement si son fibré tangent contient un sous-faisceau ample.(ii) Stabilité du fibré tangent des variétés de Fano lisses de nombre de Picard un : à l'aide de théorèmes d'annulation sur les espaces hermitiens symétriques irréductibles de type compact M, nous montrons que pour presque toute intersection complète générale dans M, le fibré tangent est stable. La même méthode nous permet de donner une réponse sur la stabilité de la restriction du fibré tangent de l'intersection complète à une hypersurface générale.(iii) Non-annulation effective pour des variétés de Fano et ses applications : nous étudions la positivité de la seconde classe de Chern des variétés de Fano lisses de nombre de Picard un. Ceci nous permet de montrer un théorème de non-annulation pour les variétés de Fano lisses de dimension n et d'indice n-3. Comme application, nous étudions la géométrie anticanonique des variétés de Fano et nous calculons les constantes de Seshadri des diviseurs anticanoniques des variétés de Fano d'indice grand.(iv) Diviseurs fondamentaux des variétés de Moishezon lisses de dimension trois et de nombre de Picard un : nous montrons l'existence d'un diviseur lisse dans le système fondamental dans certain cas particulier. / This thesis is devoted to the study of complex Fano varieties via the properties of subsheaves of the tangent bundle and the geometry of the fundamental divisor. The main results contained in this text are:(i) A generalization of Hartshorne's conjecture: a projective manifold is isomorphic to a projective space if and only if its tangent bundle contains an ample subsheaf.(ii) Stability of tangent bundles of Fano manifolds with Picard number one: by proving vanishing theorems on the irreducible Hermitian symmetric spaces of compact type M, we establish that the tangent bundles of almost all general complete intersections in M are stable. Moreover, the same method also gives an answer to the problem of stability of the restriction of the tangent bundle of a complete intersection on a general hypersurface.(iii) Effective non-vanishing for Fano varieties and its applications: we study the positivity of the second Chern class of Fano manifolds with Picard number one, this permits us to prove a non-vanishing result for n-dimensional Fano manifolds with index n-3. As an application, we study the anticanonical geometry of Fano varieties and calculate the Seshadri constants of anticanonical divisors of Fano manifolds with large index.(iv) Fundamental divisors of smooth Moishezon threefolds with Picard number one: we prove the existence of a smooth divisor in the fundamental linear system in some special cases.

Opera fantasias have been denigrated as insufficiently intellectual or serious, as derivative, as merely popular or sentimental. However, many of the perceived flaws were, if not hallmarks, at least accepted realities of Italian opera composing. Like opera itself, the opera fantasia is a popular art form, stylistically predictable yet formally flexible, based heavily on past operatic tradition and prefabricated materials. I approach opera fantasias, instrumental works that use themes from a single opera as the body of their virtuosic and flamboyant material, both historically and theoretically, concentrating on compositions written for and by woodwind-instrument performers in Italy in the second half of the nineteenth century. Important overlapping strands in my theoretical framework include the concept of virtuosity and its gradual demonization, the strong gendered overtones of individual woodwind instruments and of virtuosity, the distinct Italian context of these fantasias, the presentation and alteration of opera narratives in opera fantasias, and the technical and social development of woodwind instruments. I have uncovered a large body of compositions and composers, many of whom have not been written about in English, through archival research in Milan, Naples, Parma, Bologna, and Palermo. This reveals trends in operas used for fantasias, temporally, spatially, and between instruments, as well as further trends in the use of specific melodies. I use contemporary reviews of performances and compositions to attest to the popularity of the opera fantasia throughout the second half of the nineteenth century in Italy, including oboist Antonio Pasculli as a case study. This often overlooked genre is intimately tied to the central canon and deeply connected to its social and musical contexts. Approaching the opera fantasia as a coherent and meaningful group of works clarifies a genre that has been consciously stifled and cultural resonances that still impact music reception and performance today.

科学研究費補助金 研究種目:一般研究(C) 課題番号:04640655 研究代表者:溝口 明 研究期間:1992-1994年度