All mC5-MTases are monomeric enzymes, except M. EcoHK31I and M. AquI which are MTases composed of two poly peptides. M.EcoHK31I is a mC5-MTase which recognizes the sequence 5-YGGCCR-3' and consists of polypeptide alpha and beta, with the latter gene encoded in an alternative reading frame of the former. All of the conserved motifs in mC5-MTases can be found in polypeptide alpha, except motif IX, which is located in polypeptide beta. Both polypeptides are required for in vitro methylation. / Methylation of cytosine residues in DNA occurs in diverse organisms from bacteria to humans. In higher eukaryotic organisms cytosine-C5 methyltransferase (mC5-MTase) is the only type of DNA MTase and it plays an important role in controlling a number of cellular processes including transcription genomic imprinting and DNA repair. In bacteria, there are three types of MTases, mC4-, mC5- and mAb-, classified according to the methylation site of the DNA. MTase and its cognate restriction endonuclease (ENase) form restriction-modification system. The role of MTase is to protect the host from its own ENase digestion while the ENase acts to degrade the invasion of foreign DNA. Sequence comparison of nearly 50 bacterial mC5-MTases has shown that these enzymes share an overall common protein architecture. Ten conserved motifs (I to X), each 10 to 20 amino acids in length, have been identified, five of which are highly conserved (I, IV, VI, VIII and X). In addition, all of these enzymes have a hypervariable region lying between motifs VIII and IX. It is called the target recognition domain (TRD), and is responsible for the specificity of DNA recognition and the choice of base to be methylated. / Since both of the polypeptides alpha and beta of M.EcoHK31I are sequenced and cloned into the expression vector separately, the role of DNA recognition and subunits interaction of individual polypeptides can be studied. By electromobility shift assay, we found that polypeptides alpha and beta complex recognize specific double strand oligos substrate. Polypeptide alpha-DNA formed aggregates and polypeptide beta alone did not bind DNA. Therefore, polypeptide beta assists the proper binding of polypeptide alpha to DNA substrate. Complex of polypeptide alpha and a polypeptide beta variant with N-terminal deletion of 41 amino acids showed a 16-fold reduction in methylation activity. Further deletion resulted in an inactive MTase. By surface plasmon resonance assay, the dissociation equilibrium constant (KD) of polypeptides alpha and beta complex was found to be 56.2nM and the KD for polypeptide alpha and DeltaN46-polypeptide beta complex was increased by about 95 folds, contributing by a drastic decrease in dissociate rate constant (kd) and an increase in association rate constant (ka). This indicated that the N-terminal region of polypeptide beta takes part in subunit interaction. / To pinpoint which amino acid residues located at the variable region of polypeptide alpha are important for DNA binding and subunits interaction, "charge-to-alanine scanning mutagenesis" were performed on 16 charge residues between Asp213 and Glu271 in the small domain. It was found that the five charge residues upstream of motif X are not required for activity. For other residues except K225, E240 and D245, the protein is active when the same charge is maintained. / Fung Wai To. / "March 2006." / Adviser: P. C. Shaw. / Source: Dissertation Abstracts International, Volume: 67-11, Section: B, page: 6376. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 180-201). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

The formation of base pair radical anions and cations is closely related to many fascinating research fields in biology and chemistry such as genetic mutation, radiation-induced DNA damage and dynamics of charge transfer in DNA. However, the relevant knowledge so far mainly comes from studies on isolated base pair radical anions and cations, and their behavior in the DNA environment is less understood. In this study, we focus on how the nucleobase sequence affects the properties of the guanine¡Vcytosine (G:C) base pair radical anion and cation. The energetic barrier and reaction energy for the proton transfer along the N1(G)¡VH¡E¡E¡EN3(C) hydrogen bond and the stability of (G:C)¡E (i.e., electron affinity and ionization potential of G:C) embedded in different sequences of base-pair trimer were evaluated using density functional theory and two-layer ONIOM method. The computational results demonstrated that the presence of neighboring base pairs has an important influence on the behavior of (G:C)¡E in the gas phase. The excess electron and positive hole were found to be localized on the embedded G:C and the charge leakage to neighboring base pairs was very minor in all of the investigated sequences. Accordingly, the sequence behavior of the proton transfer reaction and the stability of (G:C)¡E is chiefly governed by electrostatic interactions with adjacent base pairs. However, the effect of base stacking, due to its electrostatic nature, is severely screened upon hydration, and thus, the sequence dependence of the properties of (G:C)¡E in aqueous environment becomes relatively weak and less than that observed in the gas phase. The effect of geometry relaxation associated with neighboring base pairs as well as the possibility of proton transfer along the N2(G)¡VH¡E¡E¡EO2(C) channel have also been investigated. The implications of the present findings to the electron transport and radiation damage of DNA are discussed.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 125-139).

Résumé: Les dommages à la cytosine, spontanés et induits par des oxydants, sont probablement la principale cause des transitions GC vers AT, la mutation du génome la plus commune chez les organismes aérobiques. Ces dommages sont impliqués dans le processus de mutagenèse, dans le vieillissement et dans le cancer. Les dommages à la cytosine par les oxydants et les radicaux libres sont nombreux et ont été découverts et étudiés dans les monomères de cytosines et dans de courts oligonucléotides. Dans cette étude, nous avons développé une méthode d'analyse par HPLC-MS/MS des plus importants produits d'oxydation de la cytosine dans l'ADN. Cette méthode permet l'analyse de la formation de 5-hydroxy-2'-désoxycytidine (5-OH-dC), de 5,6-dihydroxy-5,6-dihydro-2'-désoxyuridine (dUg), de 1-(2-désoxyribose)-5-hydroxyhydantoïne (HdU) et de 3-(2-désoxyribose)-1-carbamoy1-4,5-dihydroxy-2-oxo-imidazolidine (C422-dC) lors d'irradiation aux rayons gamma par réaction de type fenton et par ozonolyse. Les résultats de l'irradiation de l'ADN aux rayons gamma (en modifications/106bases/Gy) sont de 1.62 pour la 5-OH-dC, de 1.48 pour le dUg, de 7.43 pour la HdU et de 1.38 pour la C422-dC. La réaction de type fenton avec le cuivre donne une formation de dommages environ 25 fois plus grande qu'avec le fer et les deux types (cuivre et fer) donnent des ratios de produits semblables à ceux par les rayons gamma avec une augmentation de la 5- OH-dC et une diminution de la HdU. L'exposition .de l'ADN à l'ozone donne une très grande formation de la HdU et une faible formation des autres produits d'oxydation.//Abstract: The damages to cytosine, spontaneous and inducted by oxidants, are probably the principal cause of the GC to AT transition, the most important mutation of the genome for the aerobic organisms. Those damages are involved in the process of mutagenesis, aging and cancer. The damages to cytosine by oxidants and fre radicals are numerous and have been discovered and studied in monomers of cytosine and in short oligonucleotides. In this study, we have developed a analysis method of the most important oxidation products of cytosine in DNA by HPLC-MS/MS. This method allows the analysis of the formation of 5-hydroxy-2'-deoxycytidine (5-OH-dC), de 5,6-dihydroxy-5,6-dihydro-2'-desxyuridine (dUg), de 1-(2-deoxyribose)-5-hydroxyhydantoin (HdU) et de 3-(2-deoxyribose)- 1-carbamoyl-4,5-dihydroxy-2-oxo-imidazolidine (C422-dC) after irradiation by gamma rays, by fenton type reaction and ozonolysis. The results of the irradiation of DNA by gamma rays (in modifications10[indice supérieur 6] bases/Gy) are of 1.62 for 5-OH-dC, of 1.48 for dUg, of 7.43 for HdU and of 1.38 for C422-dC. The fenton type reaction with copper gives a formation of damages about 25 times higher than with ferrous and both kind gives a ratio of formation similar to the the ones by gamma rays with a increase of 5-OH-dC and a decrease of HdU. The exposition of DNA to ozone gives a strong formation of HdU and a small formation of the other modifications. [symboles non conformes]

Increased survival of presynaptic-like neuronal profiles was found in cell cultures of rat cerebellum when the non-neuronal cell numbers were reduced with an antimitotic drug. In both treated and untreated cell cultures, neurites grew onto the polylysine-coated surface of sepharose beads and formed a swelling. The neuronal swelling contained an accumulation of synaptic vesicles and a membrane density at the site of contact with the bead and was called an apparent presynaptic element. The apparent presynaptic elements in untreated cultures increased in number from the time the beads were added to the culture to 7 days incubation and then showed a decrease to one half the 7-day value at 14 days incubation. A 75% reduction in cell division of non-neuronal cells was seen in cultures exposed to a 5 × 10-6 M cytosine arabinoside (Ara-C) for 2 days. Adding polylysine-coated beads to cultures treated with Ara-C showed at 14 days incubation a 7-fold increase in the number of apparent presynaptic elements as compared to untreated cultures. Additional experiments examined the numbers of neurites on the beads and found only small differences between treated and untreated cultures. A decrease, however, was shown in the number of glial fibrillary acidic protein staining astrocytes on the surface of the beads in treated cultures. The reduction of astrocytes by Ara-C appeared to enhance the survival of apparent presynaptic elements but did not enhance the growth of neurites. These results suggest that proliferating non-neuronal cells at a site of injury in the central nervous system may inhibit the formation of synaptic contacts and the growth of neurites through the site of injury.

Cancer is a disease characterized by complexity and unpredictability. Consequently, its treatment is difficult and all too often unsuccessful. Almost all cancers are treated with some combination of the traditional anti-cancer armamentarium: surgery, chemotherapy, and radiotherapy. Recently, however, gene therapy has emerged as a promising addition to this existing repertoire. Its application as a single agent, or in combination with other anti-cancer treatments is proving successful in both pre-clinical and clinical settings. In this work I have investigated the combination of a conventional chemotherapy drug, cisplatin, with a type of cancer gene therapy known as cytosine deaminase + 5-fluorocytosine suicide gene therapy. Suicide gene therapy is the intracellular conversion of non-toxic prodrug to its active form by a metabolic enzyme of non-mammalian origin. There are many established enzyme/prodrug combinations, but here the bacterial enzyme cytosine dearninase (CDA) was used to convert inert 5-fluorocytosine (5FC) to highly toxic 5-fluorouracil (5FU). Of the various vector systems for therapeutic gene delivery, adenoviral (Ad) vectors have proven particularly suitable for application to cancer. This work used a first generation adenovirus type 5 vector expressing the enzyme cytosine deaminase (AdCDA) cloned from E. coli. The combination of AdCDA/5FC with cisplatin was chosen because the combination of 5FU and cisplatin, both of which are used extensively in cancer treatment, has proven effective clinically and demonstrates synergy in vitro. This combination was evaluated in murine mammary carcinoma MTIA2 cells, human colorectal carcinoma HT29 cells, HT29pl4 cells, the photofrin resistant sub-line of HT29 cells, and murine melanoma Bl6/FIO cells. The classical clonogenic assay was used to evaluate this combination treatment since it provides an accurate indication of the effectiveness a cancer treatment will have in vivo. AdCDA infected MTIA2, HT29, and HT29pl4 cell lines exhibited a dose response to increasing concentrations of SFC that was significantly different from control vector infected cells. Similarly, uninfected cells demonstrated a dose response to increasing concentrations of cisplatin. The effect of the combination on clonogenic survival, administered in the sequence of a 48 h exposure to SFC followed by 1 h exposure to cisplatin, was greater than additive compared to the effect of the two treatments alone. F10 cells exhibited a dose response to increasing concentrations of cisplatin. However, it could not be shown reproducibly that AdCDA infected FlO cells exhibited a dose response to SFC that differed significantly from control vector infected cells. Work with the FlO cells was inconclusive regarding the combination treatment, but it rendered information regarding the sensitivity of these cells to what is hypothesized to be an unidentified component present in some preparations of 5FC. Evaluation of this treatment in vivo, using both murine and human tumor cell lines, will further define the potential of AdCDA/5FC + cisplatin as a clinically relevant cancer treatment. / Thesis / Master of Science (MSc)

Many diseases are caused due to abnormalities in production of specific protein. Across this protein the conventional lock and key mechanism shows binding at the specific cites of protein. However use of antisense technology can prevent formation of protein. It does so by binding to mRNA and prevents transcription. The structural modifications lead to synthetic molecules with 18-mer units which show significant improvement in binding properties, this gives birth to a new class of oligomers called Peptide Nucleic Acid (PNA). We herein report cysteine based PNA called CPNA.

The present thesis investigates the electronic and structural properties of adenine, cytosine, and guanine layers on hydrogen passivated silicon (111)(7x7). The (7x7) reconstruction of the silicon surface was achieved by direct current heating of the samples in UHV conditions. After in situ hydrogen passivation layers of the DNA bases were prepared in different thicknesses by means of organic molecular beam deposition, all samples were characterized employing valence band and core level photoemission spectroscopy. Additionally the near edge x-ray absorption fine structure of the DNA base layers was investigated. A detailed and consistent picture of structural and electronic properties of the nucleotide bases in the solid state could be developed by comparison of measurements and DFT/B3LYP calculations.

Thesis (M.S.)--University of Florida, 2005. / Typescript. Title from title page of source document. Document formatted into pages; contains 50 pages. Includes Vita. Includes bibliographical references.

Orientador: Marcelo Rocha Marques / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba. / Made available in DSpace on 2018-08-20T06:47:39Z (GMT). No. of bitstreams: 1 Camargo_Glauciade1985-_M.pdf: 1041410 bytes, checksum: aba09e2e49414c9f9f0be4938f591a70 (MD5) Previous issue date: 2012 / Resumo: A periodontite crônica é uma doença inflamatória que leva à perda de inserção de elementos dentários, e é desencadeada e mantida por um biofilme subgengival periodontopatogênico. A presença de alguns tipos de lipopolissacarídeos (LPS), derivados de bactérias no sítio periodontal doente, pode iniciar uma sinalização por meio das células do tecido gengival, que culminará com um microambiente com diferentes células do sistema imune e com uma alteração no padrão de expressão de citocinas inflamatórias. Já foi evidenciado que no tecido gengival de pacientes com periodontite crônica, genes que codificam receptores celulares para o LPS, podem sofrer alterações epigenéticas. O objetivo deste estudo foi avaliar se a periodontite crônica e o LPS bacteriano derivado de P. gingivalis podem modular a expressão gênica de alguns fatores reguladores de eventos epigenéticos. Biópsias de tecido gengival inflamado e sem inflamação foram coleados de pacientes com periodontite crônica e de pacientes saudáveis respectivamente, o RNA total foi extraído e a expressão dos genes DNMT1 (DNA metiltransferase 1), DNAMT3a (DNA metiltransferase 3a), histona demetilase JMJD3 e histona demetilase UTX foram analisadas por meio de RT-PCR quantitativo. Fibroblastos gengivais humanos derivados de cultura primária, e queratinócitos (HaCaT) foram expostos a LPS de P. gingivalis ou ao veículo do LPS, e foram avaliadas a viabilidade celular por meio do teste MTT e a expressão gênica de DNMT1, DNMT3a, JMJD3 e UTX por meio de RT-PCR quantitativo. As análises dos resultados demonstraram que nem a periodontite e nem o LPS exposto a fibroblastos gengivais foram capazes de modular a expressão dos genes estudados. Contudo, o LPS promoveu a diminuição da expressão de DNMT1, DNMT3a e JMJD3 nas células HaCaT. Pode-se concluir que LPS derivado P. gingivalis pode modular, em queratinócitos, a expressão gênica de algumas enzimas promotoras de eventos epigenéticos / Abstract: The aim of this study was to assess whether P. gingivalis LPS can modulate, in culture of the human keratinocytes and human gingival fibroblasts, gene expression levels of the some enzymes that promote epigenetic events. In addition, the same enzymes were evaluated in sample from healthy and periodontitis affected individuals. Primary gingival fibroblast culture and keratinocytes (HaCaT) were treated with medium containing P. gingivalis LPS or P. gingivalis LPS vehicle for 24hs. After this period, cell viability were assessed by MTT test, and total RNA were extracted to evaluate gene expression levels of the enzymes: DNMT1 (DNA methyltransferase 1), DNMT3a (DNA methyltransferase 3a), histone demethylases JMJD3 and UTX, by qRT-PCR. To evaluate the gene expression in healthy and periodontitis affected individuals, total RNA was extracted from biopsies of gingival tissue from sites with (periodontitis) or without periodontitis (healthy), and gene expression of DNMT1, DNAMT3a, JMJD3 and UTX were evaluated by qRT-PCR. No significant differences were found in the gene expression analysis between healthy gingival tissues and gingival tissue from periodontitis sites. The results showed that LPS downregulated DNMT1 (p<0.05), DNMT3a (p<0.05) and JMJD3 (p<0.01) gene expression in HaCaT cells, but no modulation was found to gingival fibroblasts. P. gingivalis LPS exposure to keratinocytes, downregulates gene expression of the enzymes that promote epigenetic events / Mestrado / Histologia e Embriologia / Mestre em Biologia Buco-Dental