A poluição atmosférica vem recebendo crescente atenção como problema de saúde pública, pois representa uma fonte de agentes que podem promover o stress oxidativo e danos ao DNA, levando a efeitos cancerígenos e mutagênicos. As vias aéreas superiores, incluindo a cavidade oral são as áreas mais expostas do sistema respiratório, a material particulado e gases, e podem ser facilmente acessadas para monitorar a exposição humana a estes agentes. Nosso objetivo foi analisar o impacto da poluição do ar sobre a incidência de mutagénese em habitantes de nossa cidade. Para o estudo da mutagênese, utilizamos o teste de micronucleous (Mn), em células do epitélio da mucosa oral. Uma média de 4 a 6 amostras foram coletadas de cada voluntário, que concordou em participar do estudo, em São Paulo (SP) (n = 39) e, em Peruibe (PER), uma pequena cidade à beira-mar (n = 24), utilizados como um grupo controle. Os níveis de material particulado (PM10) foram medidos pelo método gravimétrico, e o ozônio e NO2 foram medidos pelos amostradores passivos, bem como pelas medições realizadas pela CETESB no mesmo local da coleta. Os resultados expostos em relação à concentração de MN / células, mostraram uma diferença estatisticamente significativa comparando SP (0042 ± 0032) e Per (0023 ± 0019), p = 0,009. Um aumento de MN / células foi observado em fumantes comparados aos não fumantes em SP (0055 ± 0012 e 0.040 ± 0005), p< 0,001 e em Per (0,0268 ± 0,00167 and 0,0181 ± 0,00128), p<0,001. Nosso estudo confirma os efeitos da poluição do ar na promoção de mutagênese e sugere possível sinergismo entre poluição e tabagismo / Air pollution is receiving crescent attention as a public health problem, as it represents a source of agents that may promote oxidative stress and DNA damage, leading to mutagenic and carcinogenic effects. The upper airways, including the oral cavity are the most exposed areas of the respiratory system to airborne particles and gases, and can be easily accessed to monitor human exposure to these agents. Our aim was to analyze the impact of air pollution on the incidence of mutagenesis in habitants of our city. To address mutagenicity, we used the micronucleous (mn) assay in desquamated cells of the oral mucosa. An average of 4 to 6 samples were collected from each subject that agreed to participate in the study in Sao Paulo (SP) (n=39) and in Peruibe (Per), a small city by the sea, (n=24) used as a control group. The levels of particulate (PM10) were measured by gravimetric methodology, ozone and NO2 were measured by passive monitors as well as by governmental monitoring stations in each location. The results exposed as mn/cells showed a statistically significant difference comparing SP (0,042 ± 0,032) and Per (0,023 ± 0,019), p= 0,009. An increase in mn/cells was observed in smokers compared to non smokers in SP (0,055 ± 0,012 and 0,040 ± 0,005), p<0,001 and in Peruíbe (0, 0268 ± 0,00167 and 0,0181 ± 0,00128), p<0,001. Our study confirms the hazardous effects of air pollution promoting mutagenesis and suggests that its effects may be synergic to smoking habit. We hope that our work may influence habits and public politics in order to improve public health in this issue

Neste trabalho desvendamos o papel das DNA polimerases dinB e dnaE2 em C. crescentus na mutagênese espontânea usando dois marcadores moleculares xylbla e CItet. Observamos que as taxas de mutação dos marcadores não variam significativamente entre dinB, dnaE2 e parental, coincidindo com os resultados prévios com o gene rpoB. As trocas de bases, tanto no gene cI, como em xylR, há um predomínio de mutações ATCG, como observado em rpoB, e diferente da região PxylX em xylbla. O gene xylR apresenta um hotspot que promove a inserção de uma citosina após a base 230. Neste marcador observamos que a presença de pequenas deleções (frameshifts-1) de uma base na cepa selvagem e dnaE2. Esse tipo de mutação não está presente na linhagem dinB. Esses resultados sugerem um papel importante de dinB na formação de deleções (frameshifts-1) in vivo em C. crescentus. Também observou-se que o agente 4-NQO não induz mutagênese em C. crescentus, ao contrário de E. coli. Também observamos pouca eficiência da atividade de uracila glicosilase em C. crescentus quando comparada com E. coli. / In this work we analyzed the role of DNA polymerases dinB and dnaE2 in spontaneous mutagenesis in C. crescentus, using two molecular markers: xylbla and Cltet. Our studies show that there is no significant difference in mutation rates in both markers between dinB, dnaE2 and wild type; this agrees with previous results using rpoB gene. Here, we report that there is a predominance of ATGC transitions in either cl gene or xylR, which was also shown in rpoB; however, this differs in PxylX region of xylbla. We also observed that xylR presents a mutation hotspot that promotes cytosine insertion after base 230.The presence of small one-base deletions (frameshifts-1) in wild typecbut ells, this type of mutation does not occur in the dinB strain. These results suggest an important role for dinB in the formation of deletions (frameshifts-1) in vivo in C. crescentus. We also saw that 4-NQO agent does not induce mutagenesis in C. crescentus, as it does in E. coli. Finally, the results demonstrate a poor efficiency in UDG activity in C. crescentus, when compared to E. coli.

Thesis advisor: Evan R. Kantrowitz / A full understanding of an enzyme's catalytic mechanism and a crystal structure representative of its in vivo form are powerful tools in computational drug screening and design. In the case of aspartate transcarbamoylase (ATCase), an allosteric enzyme, the mechanism and allosteric transition are still being explored. The crystallization of the ATCase mutant Asp236 to alanine, a T-state destabilized mutant, in the presence of phosphonoacetamide (PAM) by microdialysis was successful at pH 5.7. The enzyme crystallized in the R-state in the presence of only one substrate analogue. Globally the enzyme had converted to R, but the active site domains are more open than previously observed. Due to the ordered nature of the reaction, the R-state active site exists with a variety of small molecules bound at different times through out the course of the reaction. This structure shows an R-state active site with only one substrate analogue bound, and may therefore represent the R active site after catalysis has occurred and the active site is binding new substrates to perform its reaction again. Docking studies of small molecules can be conducted using this more open, emptier active site as it may be more representative of an in vivo conformation of the enzyme just before catalysis. Additionally, Arg296, previously unobserved as part of the active site, makes a hydrogen bonding interaction with the PAM molecule. The role of this residue will require further investigation. / Thesis (BS) — Boston College, 2005. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Chemistry. / Discipline: College Honors Program.

O cigarro é considerado pela Organização Mundial de Saúde como o maior agente de poluição ambiental doméstica atualmente. Muitos estudos têm analisado os efeitos do tabagismo passivo em seres humanos, mas pouco tem sido estudado em animais domésticos, assim como crianças, são os mais afetados pelo tabagismo passivo. O presente estudo busca correlacionar o exposição ao fumo passivo com a incidência de mutagênese através do teste da presença de micronúcleos nas células epiteliais da mucosa oral de cães expostos. Paralelamente, foi estudada a possível correlação desde dado com o nível de dependência à nicotina do proprietário, avaliada pelo teste de Fagerstrom. Para a avaliação da mutagênese, esfregaços da mucosa oral foram colhidos de 48 cães, 23 fumantes passivos e 25 não expostos. O número de micronúcleos por celula foi avaliado em todas as amostras. Os dados mostram que a incidência de micronúcleos foi estatisticamente maior nos animais fumantes passivos comparado com o controle (p < 0,001). Além disso, nos animais dos proprietários com escore 8 (dependência de nicotina muito alta) no Teste de Fagerstrom, a quantidade de micronúcleos foi significativamente maior do que nos animais de proprietários com escore 6 (alta dependência). Da mesma forma, nos cães de proprietários com escore 6 a incidência de micronúcleos foi estatisticamente maior que nos cães de proprietários com escore 3. Concluiu-se que existe uma associação entre a dependência da nicotina do proprietário e a frequência de alterações citogenéticas no cão exposto ao fumo passivo. Além das implicações para a saúde do animal, as observações do presente estudo podem contribuir para um estímulo à cessação do tabagismo em proprietários de animais domésticos / Cigarette smoking is considered by WHO as the major domestic air pollution agent. Many studies have addressed passive smoking and human beings but little has been studied about domestic animals. Domestic animals as well as children are dramatically affected by passive smoking. The present work tries to correlate passive smoking and micronucleus incidence in oral mucosa smears of pet dogs. In addition we studied a possible correlation between micronucleus incidence and owner´s nicotine dependence. With this purpose, owners were submitted to Fagerström questionnaire to address nicotine dependence. In the same way, oral mucosa smears were collected from 48 animals, 23 passive smokers and 25 not exposed to passive smoking. Micronucleus counts were performed in all samples at light microscopy. We observed a statistically significant difference (p < 0,001) between the two groups with an increased incidence of micronucleus formation in the passive smokers group compared to controls. Moreover, in the animals whose owners were at score 8 (very high nicotine dependence) on the Fagerstrom Test, the number of micronuclei was significantly higher than in animals with owners of score 6 (high dependency). Likewise, in dogs whose owners had a score 6 the incidence of micronuclei was statistically higher than in dogs whose owners had a score 3. It was concluded that there is an association between nicotine dependence of owners and frequency of cytogenetic alterations in dogs exposed to secondhand smoke. Besides ambient health impacts, the observations of the present study may contribute in helping dog owners quit smoking

by Rebecca McGreal. / Vita. / Thesis (Ph.D.)--Florida Atlantic University, 2012. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2012. Mode of access: World Wide Web. / The central premise of this dissertation is that the small heat shock protein (sHSP), (Sa(BB-crystallin is essential for lens and retinal pigmented epithelial (RPE) cell function and oxidative stress defense. To date, the mechanism by which it confers protection is not known. We hypothesize that these functions could occur through its ability to protect mitochondrial function in lens and RPE cells. To test this hypothesis, we examined the expression of (Sa(BB-crystallin/sHSP in lens and RPE cells, we observed its localization in the cells, we examined translocation to the mitochondria in these cells upon oxidative stress treatment, we determined its ability to form complexes with and protect cytochrome c (cyt c) against damage, and we observed its ability to preserve mitochondrial function under oxidative stress conditions in lens and RPE cells. In addition to these studies, we examined the effect of mutations of (Sa(BB-crystallin/sHSP on its cellular localization and translocation patterns under oxidative stress, its in vivo and in vitro chaperone activity, and its ability to protect cyt c against oxidation. Our data demonstrated that (Sa(BB-crystallin/sHSP is expressed at high levels in the mitochondria of lens and RPE cells and specifically translocates to the mitochondria under oxidative stress conditions. We demonstrate that (Sa(BB-crystallin/sHSP complexes with cyt c and protects it against oxidative inactivation. Finally, we demonstrate that (Sa(BB-crystallin/sHSP directly protects mitochondria against oxidative inactivation in lens and RPE cells. Since oxidative stress is a key component of lens cataract formation and age-related macular degeneration (AMD), these data provide a new paradigm for understanding the etiology of these diseases.

Submitted by admin tede (tede@pucgoias.edu.br) on 2017-05-12T13:26:04Z No. of bitstreams: 1 MARIA ALICE MONTES DE SOUSA.pdf: 3385748 bytes, checksum: 795f69f16cc6a74a1fedfa3e4c9ecfb6 (MD5) / Made available in DSpace on 2017-05-12T13:26:04Z (GMT). No. of bitstreams: 1 MARIA ALICE MONTES DE SOUSA.pdf: 3385748 bytes, checksum: 795f69f16cc6a74a1fedfa3e4c9ecfb6 (MD5) Previous issue date: 2017-03-15 / Himatanthus obovatus, popularly known as Tiborna, is a cerrado plant used in popular medicine for treating verminoses, intestinal infection, as a blood purifying, healing, analgesic, antimicrobial and anti-inflammatory. This study aimed to evaluate the angiogenic/antiangiogenic and mutagenic/antimutagenic activity of H. obovatus latex through the experimental model of the chorioallantoic membrane (MCA) assay and the Ames mutagenicity assay. The results of the angiogenesis assay indicated that the latex of H. obovatus at concentrations 1: 2, 1: 5, 1:10 and 1:20 μL showed a significant increase in the percentage area of the vascular network in the MCA, when compared to negative (Water) and inhibitor (Dexamethasone) groups. When compared to the positive control (Regederm) did not demonstrate significant difference with the latex. In the Ames assay, the latex was tested at three concentrations (1: 2, 1: 5 and 1:10 μL/plate). The results demonstrated that the latex of H. obovatus presented mutagenic action in all concentrations. The antimutagenic analysis of the latex at concentrations was compared with the positive control (sodium azide), the results of concentrations 1: 2, 1: 5 and 1:10 did not decrease a significant decrease in the number of revertant colonies (p> 0,05), But a considerable inhibition percentage was observed indicating antimutagenic action of the latex of H. obovatus. The phytochemical composition of the latex may indicate the reason for this antimutagenic effect, the presence of terpenes and iridoids. It was concluded that the latex of H. obovatus showed angiogenic activity in all concentrations tested and in the mutagenic activity in all concentrations tested. / O Himatanthus obovatus, popularmente conhecida por Tiborna, é uma planta do cerrado utilizada na medicina popular para tratamento de verminoses, infecção intestinal, como depurativo do sangue, cicatrizante, analgésico, antimicrobiano e anti-inflamatório. Este estudo teve como objetivo avaliar a atividade angiogênica/antiangiogênica e mutagênica/antimutagênica do látex de H. obovatus através do modelo experimental de ensaio na membrana corioalantóidea (MCA) e o ensaio de mutagenicidade de Ames. Os resultados do ensaio de angiogênese indicaram que o látex de H. obovatus nas concentrações 1:2, 1:5, 1:10 e 1:20 μL, apresentou um aumento significativo na área de porcentagem da rede vascular na MCA, quando comparados aos grupos controles negativo (água) e inibidor (dexametasona). Quando comparado ao controle positivo (Regederm) não demonstrou diferença significativa com o látex. No ensaio de Ames, o látex foi testado em três concentrações (1:2, 1:5 e 1:10 μL/placa). Os resultados demonstraram que o látex de H. obovatus apresentou ação mutagênica em todas concentrações. A análise antimutagênica do látex nas concentrações foram comparadas com o controle positivo (azida sódica), os resultados das concentrações 1:2, 1:5 e 1:10 não diminuiu uma diminuição significativamente no número de colônias revertentes (p>0,05), mas observou-se uma porcentagem de inibição considerável indicando ação antimutagênica do látex de H. obovatus. A composição fito-química do látex pode indicar o motivo deste efeito antimutagênico, a presença de terpenos e iridóides. Concluiu-se que o látex de H. obovatus apresentou atividade angiogênica em todas as concentrações testadas e na atividade mutagênica em todas as concentrações testadas.

Levels of the enzyme α-methylacyl-CoA racemase (AMACR) are increased ca. 9-fold in prostate cancer cells. AMACR is a very promising novel drug target as reducing AMACR levels converts castrate-resistant prostate cancer cells to androgen-dependent cells which will respond to androgen-deprivation. Despite the importance of AMACR in prostate and other cancers, there are very limited numbers of AMACR inhibitors described to-date. This is mainly due to the absence of a high-throughput assay for the screening of inhibitors against AMACR. The active-site residues and catalytic mechanism of human AMACR are still unknown, which make the rational design of drugs targeting AMACR very difficult. A range of novel potential inhibitors were synthesised using a rational drug design approach to explore the structure-activity relationship (SAR) on the side-chains of AMACR inhibitors. Their potencies were assessed using the fluoride elimination assay based on 1H and 19F NMR. Potency, mode of binding and kinetic parameters of these inhibitors were assessed using the multi-well colorimetric assay, which is the first AMACR high-throughput continuous assay reported to-date. A site-directed mutagenesis study was carried out to identify the active-site residues and catalytic mechanism of human AMACR. His-122, Asp-152, Met-184 and Glu-237 were identified as potential active-site residues, so the cDNA was mutated and expressed. The activity of wild-type and mutant AMACR enzymes were assessed using the deuterium wash-in, fluoride elimination and multi-well colorimetric assays. Results from these assays showed that human AMACR does not operate using a ‘two-base’ mechanism. Instead, it operates using a ‘one-base’ mechanism, most likely via water molecules acting as intermediaries within the hydrogen-bondings network in the active site. The knowledge obtained from this research informs rational drug design for this castrate-resistant prostate cancer target.

A glutamina e o glutamato são aminoácidos importantes no metabolismo bacteriano. A captação desses aminoácidos ocorre por meio de transportadores ativos do tipo ABC. No presente estudo avaliamos os sistemas de captação de glutamina/glutamato em Streptococcus mutans. Análises in silico revelaram a presença de dois operons que codificam sistemas de transporte de glutamina/glutamato: o operon gln, composto pelos genes e um segundo operon putativo composto pelos genes smu.1177c, smu.1178c e smu.1179c. Por meio de mutagênese sítio-específica foi possível obter a deleção do operon constituído pelos genes smu.1177c, smu.1178c e smu.1179c. O mutante (linhagem KG1) obtido apresentou redução na captação de glutamato e glutamina. O mesmo ocorreu em relação ao mutante deficiente no operon gln. O mutante KG1 apresentou maior capacidade de adesão a superfícies abióticas. Os resultados obtidos revelaram que tanto o operon gln como o operon definido pelos genes smu.1177c, smu.1178c e smu.1179c estão envolvidos na captação de glutamina e glutamato pelo S. mutans. / Glutamine and glutamate are important amino acids for bacteria metabolism. Their uptake occurs via active transporter systems of the ABC family. On the present study we evaluate the glutamine/glutamate transport systems of Streptococcus mutans. In silico analysis revealed the presence of two polycistronic operons related to transport of glutamine/glutamate: the gln operon and a putative operon composed by smu.1177c, smu.1178c and smu.1179c genes. A mutant deleted on the smu.1177c, smu.1178c and smu.1179c was successfully obtained by site-direct mutagenesis (KG1 strain), which demonstrated an impairment on glutamine and glutamate uptake, as shown by the mutant deleted on the gln operon. The KG1 strain demonstrated a gain on the abiotic surface adhesion The results revealed that both, gln operon and the operon consisted by smu.1177c, smu.1178c and smu.1179c, are involved to the internalization of glutamine and glutamate on S. mutans.

Os bancos de dados atualizados constituídos a partir de informações dos prontuários de pacientes HIV+ são importantes fontes para a realização de pesquisas clínicas e epidemiológicas de forma rápida e eficiente. A elevada variabilidade do HIV-1, resultado, entre outros fatores, da ausência de mecanismos eficientes de reparo durante os estágios da replicação viral, contribui para a emergência de cepas resistentes aos antiretrovirais. O objetivo deste trabalho é desenvolver e implementar um banco dados inteligente utilizando a Rede Neural Artificial Paraconsistente (RNAP), assentada na Lógica Paraconsistente Anotada, para auxiliar o mapeamento de informações contidas nos diversos formulários a fim de apoiar o mapeamento das informações provenientes dos diferentes registros médicos produzidos. O banco de dados será usado principalmente para apoiar o processo de decisão sobre a prescrição da terapia antiretroviral. Os resultados obtidos durante a pesquisa mostram que a técnica pode se tornar uma ferramenta promissora. / Updated databases made from information collected from HIV+ patients are important references to quickly and efficiently design clinical and epidemiologic studies. The high levels of variability of the HIV-1 virus, among other factors, the result of the absence of repair mechanisms during replication, strongly contribute to the establishment of resistance to antiretroviral therapy. The main objective of this study is the design and implementation of an inteligent database using the concept of Paraconsistent Artificial Neural Network (PANN) based on the Paraconsistent Annotated Logic, in order to support the mapping of the information coming from the different medical records produced. The database will be used primarily to support the decision process on the antiretroviral therapy prescription. Results obtained during the research show that the technique may become a promising tool.

O projeto visou aumentar a produção de celulases em fungos T. harzianum IPT 821 e T. reesei QM 9414. Esporos foram submetidos à radiação UV-C. Células das colônias mais capacitadas ao crescimento foram sucessivamente cultivadas em meio solidificado, contendo concentrações progressivamente reduzidas fonte de carbono, de modo a resultar pressão ambiental seletiva e crescente. Após esta etapa as linhagens isoladas foram cultivadas em meio composto por bagaço de cana/farelo de trigo na proporção 80/20, 60% de umidade, 30°C por 72h. Uma linhagem, originária da cepa IPT 821, apresentou atividade de 3,7 FP U/gms, 50% superior em relação à parental: 2,6 U/gms (p=0,001; p<0,05). Análises das frações enzimáticas indicaram uma diferença significativa (p=0,001; p<0,05) na atividade de xilanase: 4,7 U/gms (mutante) e 4 U/gms (parental). Ensaios de hidrólise, Avicel como substrato (1% de sólidos; w/v) indicaram um aumento de quase 70% na hidrólise em 48h, da mutante em comparação à parental (8,7% (mutante) e 4,8% (parental), concentração enzimática de 2,5 FP U/gms). / This project aimed to increase cellulase production in fungi T. harzianum IPT 821 and T. reesei QM 9414. Spores were exposed to UV-C radiation. Colonies were plated and those showing best growth were successively cultivated in plates containing increasingly stress conditions reduced concentrations of the carbon source thus creating a progressive selective pressure. After this pre-selection step isolated strains were cultivated in medium comprising sugar cane bagasse and wheat straw, in the proportion of 80/20 respectively, 60% of moisture, 30°C for 72h. One strain, originated from IPT 821 strain, showed a cellulolytic activity of 3,7 U/gdw; 50% superior to the parental strain: 2,6 U/gdw (p=0,001; p<0,05). Analysis of the enzymatic cocktail showed significant difference (p=0,001; for p <0,05) on xylanase activity: 4,7 U/gdw (mutant strain) and 4 U/gdw (parental strain). Hydrolysis assays, using Avicel as substrate (1% w/v) showed an increase on hydrolysis of about 70%, in 48h (8,7% (mutant strain) and 4,8% (parental strain), enzyme load of about 2,5 U/gdw).