Telomeres are nucleoprotein complexes found at the ends of linear eukaryotic chromosomes. Telomeres consist of a short sequence of repetitive double stranded DNA, TTAGGG repeats in humans (and all mammals), and a complex of 6 proteins, termed the shelterin complex. The length of the telomeres varies greatly between species, from approximately 300 base pairs in yeast to many 10-15 kilo bases in humans, because of the end replication problem this length get shorten with each cell division and ultimately leads to cell death. However the immortal eukaryotic cells and some transformed human cells over come this incomplete end replication problem with the use of enzyme called Telomerase. Telomerase is a ribonucleoprotein enzyme that adds a specific DNA sequence repeats (TTAGGG) to the 3¢ end of DNA strands in the telomere regions. However from the telomerase activity studies, it was concluded that telomerase is active in almost 90% of human cancers but not in normal somatic tissues. Finally, the low or transient expression of telomerase in normal tissues, including normal stem cells, and the generally longer telomeres in normal cells versus tumor cells provide a degree of tumor specificity to telomerase-based drugs and reduce the probability of toxicity to normal tissue. All of these factors suggest that cancer drugs based on telomerase might have a broad therapeutic window. This dissertation focusing on the synthesis of acridine derivatives that have the capability to inhibit the enzyme telomerase. Several N-acridyl maleimide (NAM), N-acridyl succinimide (NAS) and N-acridyl phthalimide (NAP) derivatives have been synthesized and evaluated for their anti cancer activity against various cancer cell lines. While synthesizing acridine derivatives it was required to form the C-N bonds at various stages. Developed a copper-nicotinic acid complex, which catalyzes the coupling of aryl halides with N-formyl amines and cyclic imides to form C-N bond. Explored Cu (II) catalyzed formation of C-N bond by coupling aryl halides with various N-nucleophiles such as formamide, N,N-dimethyl formamide, N-formyl amines and various cyclic imides.

A humanidade tem aumentado drasticamente o uso de antibióticos, antifúngicos, inseticidas, herbicidas e agentes quimioterápicos para tratar infecções, câncer e obter ganho econômico com a produção agrícola e industrial. O repetido uso destas substâncias leva freqüentemente à sua ineficiência devido à seleção de organismos resistentes ou tolerantes, com graves conseqüências econômicas e sociais. Os mecanismos envolvidos no processo de resistência à antifúngicos são pouco conhecidos. A compreensão destes mecanismos auxiliará no desenvolvimento de estratégias para a identificação de isolados clínicos resistentes, no tratamento de infecções fúngicas, na prevenção do surgimento de isolados resistentes, na definição de novas estratégias de utilização de antifúngicos, na revelação de novos alvos terapêuticos e portanto, no controle dos patógenos. Para o entendimento das bases moleculares da resistência à acriflavina e outros agentes inibidores em fungos nós clonamos, por transformação, um gene que confere esta resistência em Aspergillus nidulans e o caracterizamos molecularmente. Construímos uma biblioteca a partir de uma linhagem duplo-resistente e isolamos um clone que se mostrou capaz de transformar uma linhagem receptora sensível em resistente à acriflavina. A seqüência deste clone proveniente do mutante resistente, e de seu alelo selvagem revelou um gene de aproximadamente 2276 nucleotídeos traduzido em 697 aminoácidos, com alta similaridade com a trealose sintase/fosforilase (glicosiltransferase) de vários organismos. Esta seqüência foi depositada no “GenBanK" (AY102266). As enzimas trealose sintase e fosforilase participam da síntese da trealose que, além de ser fonte de carbono, está relacionada com a proteção das proteínas de membrana e das enzimas, e contra o estresse térmico e oxidativo em fungos filamentosos. As seqüências nucleotídicas dos alelos selvagem e mutante não apresentaram diferenças nas regiões estruturais ou promotoras. No entanto, a seqüência do cDNA da linhagem selvagem apresenta um íntron extra quando comparada com o cDNA da linhagem mutante. Portanto, o mRNA do gene da linhagem mutante não estaria sendo adequadamente processado, provavelmente por uma alteração no mecanismo envolvido neste processamento, inviabilizando a funcionalidade da trealose sintase / fosforilase produzida. O nocaute deste gene e a análise do fenótipo dos mutantes nulos na presença de acriflavina ou brometo de etídio confirmaram que ele não é essencial para o fungo. Através de genética clássica verificou-se que não há interação gênica ou sinergismo entre as mutações acrA1, que confere resistência à acriflavina e a outros inibidores, e tebA1, que confere resistência ao antimicótico terbinafina no fungo A. nidulans. / Mankind has drastically increased the use of antibiotics, antifungals, insecticides, herbicides and chemotherapeutic agents to treat infections and cancer and to obtain economic gains with agricultural and industrial production. The continuous use of these substances frequently leads to their inefficiency due to the selection of resistant or tolerant organisms, with serious economic and social consequences. The mechanisms involved in the process of antifungal resistance are little known. Understanding these mechanisms will help in the development of strategies for the identification of resistant clinical isolates, the treatment of fungal infections, the prevention of the occurrence of resistant isolates, the definition of new strategies for the use of antifungal agents, and the discovery of new therapeutic targets, and therefore the control of pathogens. To better understand the molecular basis of resistance to acriflavine and other inhibitory agents among fungi we cloned by transformation a gene that confers this resistance to Aspergillus nidulans and characterized it molecularly. We constructed a library from a double-resistant strain and isolated a clone that proved to be able to transform a receptor strain sensitive into an acriflavine-resistant strain. The sequence of this clone obtained from the resistant mutant and of its wild allele revealed a gene of approximately 2276 nucleotides translated into 697 amino acids, with high similarity to the trehalose synthase/phosphorylase (glycosyltransferase) of various organisms. This sequence was deposited in GenBanK (AY102266). The enzymes trehalose synthase and trehalose phosphorylase are related to the synthesis of trehalose, which in addition to being a carbon source is related to protection of the membrane proteins and of the enzymes against thermal and oxidative stress in filamentous fungi. The nucleotide sequences of the wild and mutant alleles did not show differences in the structural or promoter regions. However, the cDNA sequence of the wild strain presents an extra intron compared to the cDNA of the mutant strain. Thus, the mRNA of the gene of the mutant strain may not be adequately processed, probably due to an alteration in the mechanism involved in this processing, leading to inviability of the functionality of the trehalose synthase/phosphorylase produced. Knock out of this gene and analysis of the null mutant phenotypes in the presence of acriflavine or ethidium bromide confirmed that this gene is not essential for the fungus. Using classical Genetics, no gene interaction or synergism was observed between the acrA1 mutation, which confers resistance to acriflavine and to other inhibitors, and the tebA1 mutation, which confers resistance to the antimycotic agent terbinafine in the fungus A. nidulans.

A humanidade tem aumentado drasticamente o uso de antibióticos, antifúngicos, inseticidas, herbicidas e agentes quimioterápicos para tratar infecções, câncer e obter ganho econômico com a produção agrícola e industrial. O repetido uso destas substâncias leva freqüentemente à sua ineficiência devido à seleção de organismos resistentes ou tolerantes, com graves conseqüências econômicas e sociais. Os mecanismos envolvidos no processo de resistência à antifúngicos são pouco conhecidos. A compreensão destes mecanismos auxiliará no desenvolvimento de estratégias para a identificação de isolados clínicos resistentes, no tratamento de infecções fúngicas, na prevenção do surgimento de isolados resistentes, na definição de novas estratégias de utilização de antifúngicos, na revelação de novos alvos terapêuticos e portanto, no controle dos patógenos. Para o entendimento das bases moleculares da resistência à acriflavina e outros agentes inibidores em fungos nós clonamos, por transformação, um gene que confere esta resistência em Aspergillus nidulans e o caracterizamos molecularmente. Construímos uma biblioteca a partir de uma linhagem duplo-resistente e isolamos um clone que se mostrou capaz de transformar uma linhagem receptora sensível em resistente à acriflavina. A seqüência deste clone proveniente do mutante resistente, e de seu alelo selvagem revelou um gene de aproximadamente 2276 nucleotídeos traduzido em 697 aminoácidos, com alta similaridade com a trealose sintase/fosforilase (glicosiltransferase) de vários organismos. Esta seqüência foi depositada no “GenBanK” (AY102266). As enzimas trealose sintase e fosforilase participam da síntese da trealose que, além de ser fonte de carbono, está relacionada com a proteção das proteínas de membrana e das enzimas, e contra o estresse térmico e oxidativo em fungos filamentosos. As seqüências nucleotídicas dos alelos selvagem e mutante não apresentaram diferenças nas regiões estruturais ou promotoras. No entanto, a seqüência do cDNA da linhagem selvagem apresenta um íntron extra quando comparada com o cDNA da linhagem mutante. Portanto, o mRNA do gene da linhagem mutante não estaria sendo adequadamente processado, provavelmente por uma alteração no mecanismo envolvido neste processamento, inviabilizando a funcionalidade da trealose sintase / fosforilase produzida. O nocaute deste gene e a análise do fenótipo dos mutantes nulos na presença de acriflavina ou brometo de etídio confirmaram que ele não é essencial para o fungo. Através de genética clássica verificou-se que não há interação gênica ou sinergismo entre as mutações acrA1, que confere resistência à acriflavina e a outros inibidores, e tebA1, que confere resistência ao antimicótico terbinafina no fungo A. nidulans. / Mankind has drastically increased the use of antibiotics, antifungals, insecticides, herbicides and chemotherapeutic agents to treat infections and cancer and to obtain economic gains with agricultural and industrial production. The continuous use of these substances frequently leads to their inefficiency due to the selection of resistant or tolerant organisms, with serious economic and social consequences. The mechanisms involved in the process of antifungal resistance are little known. Understanding these mechanisms will help in the development of strategies for the identification of resistant clinical isolates, the treatment of fungal infections, the prevention of the occurrence of resistant isolates, the definition of new strategies for the use of antifungal agents, and the discovery of new therapeutic targets, and therefore the control of pathogens. To better understand the molecular basis of resistance to acriflavine and other inhibitory agents among fungi we cloned by transformation a gene that confers this resistance to Aspergillus nidulans and characterized it molecularly. We constructed a library from a double-resistant strain and isolated a clone that proved to be able to transform a receptor strain sensitive into an acriflavine-resistant strain. The sequence of this clone obtained from the resistant mutant and of its wild allele revealed a gene of approximately 2276 nucleotides translated into 697 amino acids, with high similarity to the trehalose synthase/phosphorylase (glycosyltransferase) of various organisms. This sequence was deposited in GenBanK (AY102266). The enzymes trehalose synthase and trehalose phosphorylase are related to the synthesis of trehalose, which in addition to being a carbon source is related to protection of the membrane proteins and of the enzymes against thermal and oxidative stress in filamentous fungi. The nucleotide sequences of the wild and mutant alleles did not show differences in the structural or promoter regions. However, the cDNA sequence of the wild strain presents an extra intron compared to the cDNA of the mutant strain. Thus, the mRNA of the gene of the mutant strain may not be adequately processed, probably due to an alteration in the mechanism involved in this processing, leading to inviability of the functionality of the trehalose synthase/phosphorylase produced. Knock out of this gene and analysis of the null mutant phenotypes in the presence of acriflavine or ethidium bromide confirmed that this gene is not essential for the fungus. Using classical Genetics, no gene interaction or synergism was observed between the acrA1 mutation, which confers resistance to acriflavine and to other inhibitors, and the tebA1 mutation, which confers resistance to the antimycotic agent terbinafine in the fungus A. nidulans.

Research efforts to determine the causes, effects and locations of mutations within the human genome have been widely pursued due to their role in the development of various diseases. The main cause of mutations in vivo is oxidative damage to DNA via oxidants and free radical species. Numerous studies have been performed in vitro to determine how oxidative damage is induced in DNA. Most of these in vitro studies require photosensitizers to initiate the oxidative damage through various mechanisms. For the purposes of this research, all the photosensitizers that were used initiated oxidative damage in DNA through the electron transfer mechanism. In the charge transport studies, an anthraquinone photosensitizer was covalently linked to the 5 end of DNA by a short carbon tether in order to determine the pattern of damage induced along the length of the DNA. Anthraquinone preferentially damages guanine bases. Our first work sought to determine the effects of charge transport through guanine rich quadruplex DNA dimers. The dimers were formed by the combination of two hairpins with duplex overhangs extending beyond the quadruplex region. This enabled the optimal comparison of the effects of charge transport between duplex and quadruplex DNA structures. Another area of research we pursued in this area was to determine the effects of charge transport in M-DNA (a novel DNA conformation that was reported to form in the presence of zinc ions at a pH above 8). Earlier work on M-DNA suggested that it behaved like a molecular wire. Our research attempted to determine the effects of charge transport on this structure in order to show the behavior of a DNA molecular wire as compared to the standard studies performed in this area on normal B-DNA structures. Lastly, in collaboration with Dr. Ramaiah and colleagues we designed some viologen linked acridine photosensitizers which were tested for any ability to cleave GGG bulges. In preliminary studies, these viologen linked acridine derivatives showed preferential cleavage for guanine bases. They were not covalently bound to DNA, although they could potentially form non covalent interactions with DNA such as intercalation and/or groove binding. Our overall research goal was to determine the extent and overall effect of oxidative damage (using different photosensitizers) on the various DNA structures mentioned above.