­ì¤l¤OÅã·LÃè(AFM)¯à¦b®ð¬Û¡B¯uªÅ¡B¤Î±µªñ¥Í²z±ø¥óªº²G¬Û¤¤ª½±µ¶i¦æªí­±³æ¤À¤lªºÆ[´ú¡C¦ý¼Ë«~¤À¤l³Q©T©w«á¡A¨äµ²ºc¬O§_»P¦ÛµMª¬ºA¬ÛÃö¡A»á¥O¤H½èºÃ¡C ¥»¬ã¨s°w¹ï¶³¥À¤ùªí­±¶i¦æ¤Æ¾Ç­×¹¢¡A§Q¥ÎºëÓi(spermine)¤j¤j´£°ª¤F°òªOªí­±§lªþDNAªº¯à¤O¡C¹B¥Î»E¦X¤À¤lÃì²Î­p¤ÀªR²z½×(statistical polymer chain analysis)¡A¥H¤TºØ¤£¦Pªø«×ªº½u«¬DNA¤À¤l¡A®Ú¾ÚAFM¼v¹³¤À§O§@¤À¤l½ü¹øÁ`ªø(contour length, L)¤Î¥¼ºÝ¨âÂI¶ZÂ÷(end-to-end distance, R)ªº´ú¶q¡A¥H<R2>»PL¤§¬ÛÃö©Ê±Àª¾¼v¹³¤¤ªºDNA¤À¤lªí²{ªº¬O3D¥ßÅé®·®»ºAºc«¬(three-dimensional trapped configuration)¡A¦Ó«D¤À¤l¦b2DªÅ¶¡­«·s«Ø¥ß¥­¿Å«áªºµ²ºc¡C¥t¥~¡AÂǧïÅÜDNA¼Ë«~²Gºw¦b¶³¥À¤ù¤W°®Àꪺ®É¶¡¡A©Ò³y¦¨°òªO§lªþ¤À¤l¼Æ¥ØªºÅܤơA°t¦X¤£¥i°fÂX´²¹B°Ê¼Ò«¬±o¨ì¤F¤À¤l¥Ñ²G¬Û¨ì¹Fªí­±¿é°e¹Lµ{¤§ÂX´²«Y¼Æ¡C ¦b©Ý¾ë²§ºc酶(topoisomerase)»P¶Ê¤ÆDNA¤À¤l¤ÏÀ³¹êÅ礤¡AAFM©úÅã¿ëÃÑ¥XDNA¤À¤l¦b©Ý¾ëºc§Î¤WªºÂà¤Æ¡A¬Æ¦Üª½±µ¬Ý¨ì¸Ñ±Û¾÷¨î¤¤©Ý¾ë酶¤À¤l»PÂùªÑDNA¤¤¤@±ø³æªÑªº§@¥Î¡C

Exposure to benzene, a ubiquitous environmental pollutant, has been linked to leukemogenesis, although the mechanism of benzene initiated carcinogenesis remains unclear. It has been proposed that benzene can be bioactivated to toxic metabolites such as 1,4 benzoquinone (BQ), which can alter signalling pathways and affect chromosomal integrity. BQ has been shown to increase the activity of c-Myb, which is an important transcription factor involved in hematopoiesis, cell proliferation, and cell differentiation. The c-Myb protein also increases topoisomerase IIα (topo IIα) promoter activity specifically in cell lines with hematopoietic origin. Topo II is a critical nuclear enzyme that removes torsional strain by cleaving, untangling and religating double-stranded DNA. Since topo II mediates DNA strand breaks, aberrant topo II activity or increased protein levels may increase the formation of DNA strand breaks, leaving the cell susceptible to mutational events. I hypothesize that BQ increases c-Myb activity, which in turn increases topo IIα promoter activity resulting in increased DNA strand breaks. Using luciferase reporter assays in K-562 cells (human chronic myeloid leukemic cells) I confirmed that BQ exposure (25 and 37 µM) caused an increase in c-Myb activity after 24 hours. Contradictory to previous findings, overexpression of exogenous c-Myb or a polypeptide consisting of c-Myb’s DNA binding domain (DBD), which competitively inhibits the binding of endogenous c-Myb to DNA, did not affect topo IIα promoter activity. However, BQ exposure (37 µM for 24 hours) caused a significant increase in topo IIα promoter activity, which could be blocked by the overexpression of the DBD polypeptide. Western immunoblotting analysis did not show any significant increases in topo IIα protein levels in cells exposed to 37 µM BQ for 24 hours. Overall, this study suggests that BQ exposure increases topo IIα promoter activity through the c-Myb signalling pathway and furthers our understanding of BQ-mediated toxicity. / Thesis (Master, Pharmacology & Toxicology) -- Queen's University, 2008-01-02 14:09:00.011

Bloom syndrome is a rare autosomal recessive disorder that is caused by mutations in the BLM gene. BLM associates with TopoIIIα and RMI1 to form a complex that is essential to maintain genome integrity. This complex catalyzes a dissolution reaction that resolves recombination intermediates containing two Holliday junctions without crossing over of genetic material. Dissolution activity is remarkable because it accounts for the in vivo role of BLM-TopoIIIα-RMI1 in suppressing sister chromatid exchanges. To further understand the biochemical roles that each member of the BLM complex plays in dissolution, I generated single-stranded catenanes that resemble the proposed intermediates at the latest steps of dissolution. Using this substrate, I demonstrated that TopoIIIα is a single-stranded DNA decatenase that is specifically stimulated by BLM and RMI1. Interaction between TopoIIIα and RMI1 is essential for the optimal decatenase activity. Furthermore, binding of RPA to single-stranded DNA substrate inhibits TopoIIIα decatenase activity. However, complex formation between BLM, TopoIIIα and RMI enables TopoIIIα to displace RPA and catalyze decatenation. Since the decatenase activity is presumed to be involved in many aspects of DNA metabolism, I investigated the roles of RMI1 and TopoIIIα in DNA replication in vivo. Using the molecular combing technique, I showed that RMI1 functions downstream of BLM to promote normal replication fork progression. In addition, BLM, TopoIIIα and RMI1 colocalize with one another in response to replication stress. Finally, interaction between TopoIIIα and RMI1 is essential for nuclear localization of the complex and for the complex to promote recovery from replication stress. This work defines molecular functions for RMI1 and TopoIIIα in DNA replication and repair, providing insight into their roles as suppressors of genome instability.

Thesis (Ph. D.)--University of Texas at Austin, 1998. / Vita. Includes bibliographical references (leaves 213-221). Available also in a digital version from Dissertation Abstracts.

Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2005. / Includes bibliographical references (leaves 103-116). Also available in electronic version.

Thesis (Ed. D.)--Illinois State University, 1990. / Title from title page screen, viewed November 28, 2005. Dissertation Committee: Herman E. Brockman (chair), Alan J. Katz, Lynne A. Lucher, Radheshyam K. Jayaswal, David F. Weber, Anthony E. Liberta. Includes bibliographical references (leaves 114-131) and abstract. Also available in print.

Made available in DSpace on 2016-08-29T15:35:33Z (GMT). No. of bitstreams: 1 tese_6737_Maicon Delarmelina.pdf: 47315 bytes, checksum: 71d6ef49e51bffadf876359d21c01ed0 (MD5) Previous issue date: 2013-08-30 / O câncer é uma das doenças que mais causam temor na sociedade, por ter se tornado um estigma de mortalidade e dor. Estatisticamente, em pesquisa realizada pela Organização Mundial de Saúde (WHO), o câncer é a segunda causa de óbitos no mundo com 13%, matando cerca de 6,0 milhões de pessoas por ano. Acredita-se que em 2015 o câncer seja responsável por 9 milhões de mortes no mundo. Os avanços verificados nas últimas décadas, na área da quimioterapia antineoplásica, têm facilitado consideravelmente a aplicação de outros tipos de tratamento de câncer e permitido maior número de curas. Entretanto, muitos tipos de câncer são intrinsecamente resistentes a quimioterápicos, enquanto outros inicialmente respondem ao tratamento, porém adquirem resistência aos fármacos. A resistência múltipla ao fármaco (MDR), o quadro mais grave, ocorre quando culturas de células in vitro e in vivo mostram resistência simultânea a diferentes fármacos. Portanto, apesar do grande número de compostos que compõem o arsenal quimioterápico de combate ao câncer, o problema de resistência requer a busca constante por novos fármacos eficazes no combate ao câncer, doença que afeta milhões de pessoas. Em estudos farmacológicos com diversas quinonas foi evidenciado que esta classe de substância mostram variadas atividades farmacodinâmicas, dentre elas, as propriedades citotóxicas e inibidoras de sistemas celulares reparadores, processos nos quais atuam de diferentes formas. Como exemplo, destaca-se o estresse oxidativo que provocam ao induzirem a formação deletéria endógena de espécies bioativas derivadas do oxigênio (ROS). Outra atividade marcante destas substâncias, descoberta recentemente, é a inibição da enzima topoisomerase II, ação que provoca o desencadeamento da apoptose celular. Neste trabalho foram sintetizados em rendimentos que variaram de 52 a 89% onze derivados 1,4-naftoquinônicos contendo grupos oxigenados, nitrogenados e sulfurados nas posições 2 e/ou 3 do núcleo naftoquinônico. Posteriormente estes compostos tiveram as suas citotoxidades avaliadas nas linhagens de câncer humano de pulmão (H460), mama triplo-negativo (MDA-MB-231) e ovário (A2780). Os compostos 25f e 44, 25c e 44, e 25g e 44 apresentaram uma significativa atividade anticâncer in vitro para as linhagens de câncer de pulmão H460, mama triplo-negativos MDA-MB-231 e ovário A2780, respectivamente, demostrando um grande potencial como compostos protótipos para o desenvolvimento de novos agentes antineoplásicos. Efetuou-se também estudos de docking dos quatro compostos mais ativos com os alvos terapêuticos PI3K e topoisomerase II. Os estudos mostraram que um possível alvo terapêutico das subtâncias sintetizadas é a enzima topoisomerase II, resultado das interações estabilizadoras observadas no complexo enzima/ligante. / Cancer is one of the most feared disease in society, which have become a stigma of death and pain. Statistically, a survey conducted by the World Health Organization (WHO), cancer is the second cause of death in the world with 13%, killing about 6.0 million people per year. It is believed that in 2015 cancer will be responsible for over 9 million deaths worldwide. The advances achieved in recent decades in cancer chemotherapy research have greatly facilitated the implementation of other types of cancer treatment and allowed a greater number of cures. However, many cancers are intrinsically resistant to chemotherapy, while others respond to initial treatment, but it acquires drug resistance. The multiple drug resistance (MDR), the most severe case, occurs when in vitro and in vivo cell cultures show simultaneous resistance to different drugs. Therefore, despite the large number of compounds that make up the arsenal of anti-cancer chemotherapy, the resistance problem requires a constant search for effective new drugs in fighting cancer, a disease that affects millions of people. In pharmacological studies with various quinones was evidenced that these substances show different pharmacodynamic activities, among them, cytotoxic properties and inhibiting cellular systems repairers, processes in which they act in different ways. As an example, there is oxidative stress that cause deleterious by inducing formation of endogenous bioactive oxygen species derived (ROS). Another remarkable activity of these substances, recently discovered, is the inhibition of the enzyme topoisomerase II, an action that triggers the onset of cellular apoptosis. In this work were synthesized eleven 1,4-naphthoquinone derivatives containing functional groups, nitrogen and sulfur in positions 2 and/or 3 naphthoquinone core in yields ranging 52-89%. Subsequently these compounds had their cytotoxicity evaluated in strains of human lung cancer (H460), triple-negative breast (MDA-MB- 231) and ovarian (A2780). Compounds 25f and 44, 44 and 25c, and 25g and 44 showed a significant anticancer in vitro activity for the strains of lung cancer H460, triple-negative breast MDA-MB-231 and ovarian A2780, respectively, showing a great potential as lead compounds for the development of new anticancer agents. Docking studies of the four most active compounds with therapeutic targets PI3K and topoisomerase II were performed. Those studies have shown that a 16 possible therapeutic target of synthesized compounds is topoisomerase II enzyme because of the observed enzyme/binder complex stabilizing interactions.

Escherichia coli topoisomerase I (TopA), a regulator of global and local DNA supercoiling, is modified by Nε-Lysine acetylation. The sirtuin protein deacetylase CobB can reverse both enzymatic and non-enzymatic lysine acetylation modifications. Here, we explored the effect of lysine acetylation on E. coli topoisomerase I through analysis of TopA relaxation activity and protein expression in cell extract of wild-type and a ΔcobB mutant strains. We showed that the absence of deacetylase CobB in a ΔcobB mutant reduced intracellular TopA relaxation activity while elevating TopA expression and topA gene transcripts levels. Acetyl phosphate mediated lysine acetylation decreased the activity of purified TopA in vitro, and the interaction with purified CobB protected TopA from such inactivation. We explored the physiological significance of TopA acetylation on DNA supercoiling by two-dimensional gel analysis and on cell growth rate by growth curve analysis. We found that the absence of CobB increased negative DNA supercoiling. The slow growth phenotype of the ∆cobB mutant can be partially compensated by overexpression of recombinant TopA. In addition, the specific activity of TopA expressed from His-tagged fusion construct in the chromosome was inversely proportional to the degree of in vivo lysine acetylation during growth transition and growth arrest. Investigation of TopA relaxation mechanism using nuclease footprinting and TopA oxidative crosslinking suggested the potential association of TopA acetylation in catalysis. Mass spectrometry analysis of in vitro acetyl phosphate acetylated TopA identified abundant lysine acetylation sites. Substitution of lysine residues by site-directed mutagenesis was used to model the effect of acetylation on individual lysine residues. Our results showed that substitution of Lys-484 with alanine reduced the relaxation activity, suggesting the reduction of TopA relaxation activity by acetylation was probably in part due to acetylation on Lys-484. These findings demonstrate that E. coli topoisomerase I is modulated by lysine acetylation and the prevention of TopA inactivation from excess lysine acetylation and consequent increase in negative DNA supercoiling is an important physiological function of the sirtuin deacetylase CobB.

O cÃncer coloretal (CRC) à a 3 neoplasia mais prevalente no mundo. O irinotecano (IRI), fÃrmaco de primeira linha para os tratamentos do CRC e sua metÃstase hepÃtica, tem aumentado a sobrevivÃncia dos pacientes. Contudo, seus efeitos colaterais, incluindo a esteato-hepatite nÃo alcoÃlica (NASH), podem limitar o curso do tratamento. Os protocolos baseados em irinotecano foram associados com um aumento no risco de NASH de 3,4 vezes. A silimarina (SIL) tem mostrado ser capaz de prevenir doenÃas do fÃgado gorduroso no contexto clÃnico e em modelos de danos hepÃticos induzidos quimicamente. Assim, nosso objetivo foi estudar o efeito da SIL na NASH induzida pelo IRI, assim como o mecanismo envolvido. MÃtodos e Resultados: Camundongos Swiss fÃmeas (n=8-10), foram divididos em 6 grupos e injetados com salina (SAL 5ml/kg i.p.), IRI (50 mg/kg i.p.), SIL (150 mg/kg v.o.) ou IRI (50 mg/kg i.p.) + SIL (SIL 1,5, 15, 150 mg/kg v.o.) 3x/semana/7 semanas. Amostras de sangue foram coletadas na sÃtima semana para determinar a concentraÃÃo sÃrica das enzimas hepÃticas ALT e AST (U/L). Animais foram mortos para a coleta do fÃgado para avaliar do dano tecidual (escores de Kleiner), dosagem de lipÃdeos totais (mg/g de tecido), MDA (nmol/g tecido), NPSH (mg de NPSH/g tecido), IL-6 (pg/mg de tecido), IL-10 (pg/mg de proteÃna) e IL-1&#946; (pg/mg de tecido), imunomarcaÃÃo de Ãxido nÃtrico sintase induzida (iNOS), 3-Nitrotirosina (Ntyr), e Receptor Toll Like tipo 4 (TLR4), quantificaÃÃo do fator nuclear kappa B (NF&#954;B) e da &#945;-actina de mÃsculo liso (&#945;-SMA) e expressÃo do gene RSS. ANOVA/Teste de Newman-Keuls ou Kruskal Wallis/ Teste de Dunn foram utilizados para anÃlise estatÃstica. Foram consideradas diferentes amostras onde o nÃvel descritivo era inferior a 5%. O trabalho foi aprovado pelo comità de Ãtica em pesquisa animal sob o nÃmero de protocolo: 21/12. O IRI aumentou de forma significante as transaminases hepÃticas, o infiltrado neutrofÃlico, o acÃmo de lipÃdeos, o acÃmulo de MDA, a expressÃo de NTyr, a expressÃo de &#945;-SMA, a expressÃo de NF&#954;B, a expressÃo de IL-1&#946;, a expressÃo de IL-6, a expressÃo de TLR4 e quantificaÃÃo de DNA bacteriano, quando comparados ao grupo SAL. SIL (1,5 mg/kg) melhorou esses parÃmetros, exceto a infiltraÃÃo neutrofÃlica e a quantificaÃÃo do DNA bacteriano quando comparados ao o grupo IRI (P<0,05). Por outro lado, a dose media de SIL (15 mg/kg) foi efetiva apenas no Infiltrado NeutrofÃlico, na expressÃo de NTyr, na expressÃo de NF&#954;B e na expressÃo de IL-6. Adicionalmente, essa dose aumentou a expressÃo hepÃtica de IL-10, a quantificaÃÃo de DNA bacteriano e a expressÃo da &#945;-SMA quando comparados com o grupo IRI (p<0,05). Contudo, a expressÃo de iNOS nÃo foi afetada pelo prÃ-tratamento com SIL (P<0,05) e a maior dose foi ainda mais deletÃria. ConclusÃes: O prÃ-tratamento com SIL previne o dano hepÃtico causado pelo IRI provavelmente atravÃs da mudanÃa da resposta inflamatÃria mediada por receptores TLR4 e citocinas IL-1, IL-6, IL-10 e NF&#954;B. O dano observado no grupo de animais tratados com as maiores doses de SIL parece ser dependente da translocaÃÃo bacteriana do intestido que à associada a ativaÃÃo do TLR4. Adicionalmente, a silimarina contribui para hepatoproteÃÃo por inibir o estresse oxidativo e a nitrosilaÃÃo proteica, prevenindo a ativaÃÃo de mecanismos de fibrose hepÃtica

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Acridinas sÃo molÃculas policÃclicas aromÃticas planares que possuem a capacidade de intercalar no DNA nuclear. Muitos dos seus representantes apresentam propriedades antibacterianas, antiparasitÃrias e antitumorais. O presente estudo avaliou o potencial citotÃxico de 22 novos compostos acridÃnicos em linhagens de cÃlulas tumorais humanas. Dentre esses, quatro compostos [5-(acridin-9-il-metileno)-3-(4-metil-benzil)-tiazolidina-2,4-diona, AC4; 5-(acridin-9-il-metileno)-3-(4-bromo-benzil)-tiazolidina-2,4-diona, AC7; 5-(acridin-9-il-metileno)-3-(4-cloro-benzil)-tiazolidina-2,4-diona, AC10; e 5-(acridin-9-il-metileno)-3-(4-flÃor-benzil)-tiazolidina-2,4-diona, AC23] foram ativos, especialmente em HCT-8 (cÃlon) e SF-296 (glioblastoma), com valores de CI50 variando de 2,3 a 5,3 Âg/mL. Os compostos apresentaram seletividade para cÃlulas tumorais, desde que nÃo inibiram (IC50 > 25 Âg/mL) a proliferaÃÃo de cÃlulas monocucleares de sangue perifÃrico humano (CMSPH), bem como, nÃo foram capazes de induzir dano ao DNA dessas cÃlulas. Nenhum dos compostos mostrou atividade hemolÃtica contra eritrÃcitos de camundongos (EC50 > 200 Âg/mL), o que sugere uma citotoxicidade por mecanismos mais especÃficos. A fim de determinar o mecanismo envolvido na citotoxicidade seletiva dos compostos, foi realizada uma seqÃÃncia de experimentos in vitro, usando a linhagem HCT-8 como modelo. As cÃlulas foram tratadas em diferentes concentraÃÃes dos compostos (2,5; 5 e 10 Âg/mL) por 12 e 24 horas. Todos os compostos foram capazes de reduzir a viabilidade (teste do azul de tripan) e a proliferaÃÃo (ensaio do BrdU) de cÃlulas HCT-8 apÃs o tratamento. A induÃÃo de apoptose pelos derivados acridÃnicos foi determinada por citometria de fluxo (integridade da membrana, fragmentaÃÃo do DNA internucleosomal e potencial transmembrÃnico) e por anÃlise morfolÃgica das alteraÃÃes celulares (brometo de etÃdeo/laranja de acridina e hematoxilina/eosina). A anÃlise por citometria de fluxo revelou que os compostos avaliados promoveram despolarizaÃÃo mitocondrial, o qual evidencia a ativaÃÃo da apoptose pela via intrÃnseca nas cÃlulas HCT-8. Na anÃlise do screening em 3 diferentes linhagens mutantes de Saccharomyces cerevisiae, foi observado que a linhagem Top1&#916; (sem topoisomerase I) mostrou moderada resistÃncia aos compostos acridÃnicos testados na concentraÃÃo de 50 Âg/mL. AlÃm disso, as acridinas inibiram parcialmente o relaxamento do DNA por topoisomerase I, sugerindo que os compostos AC4, AC7, AC10, AC23 tem o potencial antiproliferativo, em parte, relacionado a inibiÃÃo da atividade catalÃtica desta enzima. Esses dados apontam o potencial anticÃncer dos compostos testados. / Acridines are planar aromatic polycyclic molecules that have the ability to progress in nuclear DNA. Many of its representatives have antibacterial, antiparasitic and antitumor. This study evaluated the cytotoxic potential of 22 new acridine compounds in strains of human tumor cells. Among these, four compounds [5-(acridin-9-yl-methilene)-3-(4-methyl-benzyl)-thiazolidine-2,4-dione, AC4; 5-(acridin-9-yl-methilene)-3-(4-bromine-benzyl)-thiazolidine-2,4-dione, AC7; 5-(acridin-9-yl-methilene)-3-(4-chloro-benzyl)-thiazolidine-2,4-dione, AC10; and 5-(acridin-9-yl-methilene)-3-(4-fluor-benzyl)-thiazolidine-2,4-dione, AC23] were active, especially in HCT-8 (colon) and SF-296 (glioblastoma), with IC50 values ranging from 2.3 to 5.3 mg / mL. The compounds were selective for tumor cells, provided they do not inhibit (IC50 > 25 Âg/mL) cell proliferation monocucleares human peripheral blood (CMSPH) and were not able to induce DNA damage to these cells. None of the compounds showed hemolytic activity against erythrocytes of mice (EC50 > 200 Âg/mL), suggesting a cytotoxicity by more specific mechanisms. In order to determine the mechanism involved in the selective cytotoxicity of compounds was carried out a sequence of in vitro experiments, using the line HCT-8 as a model. The cells were treated in different concentrations of compounds (2.5, 5 and 10 Âg/mL) for 12 and 24 hours. All compounds were able to reduce the viability test (trypan blue) and proliferation (BrdU assay) of HCT-8 cells after treatment. Induction of apoptosis by acridine derivatives was determined by flow cytometry (membrane integrity, DNA fragmentation and internucleosomal transmembrane potential) and morphological analysis of cellular changes (ethidium bromide/acridine orange, and hematoxylin-eosin). The analysis by flow cytometry showed that the compounds evaluated promoted mitochondrial depolarization, which shows the activation of apoptosis by intrinsic pathway in HCT-8 cells. In the analysis of screening in 3 different mutant strains of Saccharomyces cerevisiae, it was observed that the line Top1&#916; (without topoisomerase I) showed resistance to acridine compounds tested at a concentration of 50 Âg/mL. In addition, the acridines partially inhibited the relaxation of DNA by topoisomerase I, suggesting that the compounds AC4, AC7, AC10, AC23 has the potential antiproliferative partly related to inhibition of catalytic activity of this enzyme. These data indicate the potential anticancer compounds tested.