Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The use of treated domestic wastewater for irrigation could become an alternative to regions facing water scarcity, as in northeastern Brazil, specifically in the state of Sergipe. This work aimed to analyze the influence of the use of domestic wastewater treated in the microbiological quality of carrots (Daucus carota) and beets (Beta vulgaris) irrigated with this water. The experiment was conducted in a greenhouse of the Department of Agricultural Engineering (DEA) in the Federal University of Sergipe / Campus of Saint Kitts from April to July 2014. The wastewater was collected in the Sewage Treatment Plant (WWTP ) Rosa Elze, in the municipality of St. Kitts / SE. Irrigation was performed using four different dilutions. The evapotranspiration was obtained by the method of the Food and Agriculture Organization of United Nations (FAO) 56 through weather station mounted inside the greenhouse. The experimental design was randomized blocks (DBC), composed of eight treatments, two species and four wastewater percentage e/ou drinking water of Sergipe Supply Company (DESO); Of DESO 100% water; 75% water DESO + 25% waste water; 75% wastewater + 25% water DESO and 100% wastewater in five replicates and four plants per working portion. Microbiological samples were analyzed according to the parameters recommended by Resolution no. 12 02/01/2001 of the National Health Surveillance Agency - ANVISA: enumeration of fecal coliforms and Salmonella sp. In addition to these analyzes, they were also listed the population of mesophilic aerobic bacteria and molds and yeasts and the results were submitted to ANOVA by the Tukey test at 5% probability. The results obtained in the microbiological quality analyzes show that the beets and carrots irrigated with wastewater are in accordance with the standards established by law (Salmonella sp. Absence in 25 g fecal coliform ≤3 NMP. g-1) and the enumeration of mesophilic and molds and yeasts did not exceed 4,5x106, but we still need more depth to the thematic studies. / A utilização de águas residuárias domésticas tratadas para irrigação pode se tornar uma alternativa para regiões que enfrentam escassez de água, como na região Nordeste do Brasil, especificamente no Estado de Sergipe. A presente dissertação teve como objetivo analisar a influência do uso de águas residuárias domésticas tratadas na qualidade microbiológica de cenouras (Daucus carota) e beterrabas (Beta vulgaris) irrigadas com essa água. O experimento foi realizado em casa de vegetação do Departamento de Engenharia Agronômica (DEA), localizada na Universidade Federal de Sergipe/Campus de São Cristóvão no período de abril a julho de 2014. As águas residuárias foram coletadas na Estação de Tratamento de Esgotos (ETE) Rosa Elze, localizada no Município de São Cristóvão/SE. As irrigações foram feitas utilizando-se quatro diluições diferentes. A evapotranspiração foi obtida pelo método do Food and Agriculture Organization of United Nations (FAO) 56 através de estação meteorológica montada dentro da casa de vegetação. O delineamento experimental utilizado foi o de blocos casualizados (DBC), composto por oito tratamentos, sendo duas espécies e quatro percentuais de água residuária e/ou água potável da Companhia de Abastecimento de Sergipe (DESO); 100% água da DESO; 75% água da DESO + 25% água residuária; 75% água residuária + 25% água da DESO e 100% água residuária, em cinco repetições e quatro plantas por parcela útil. As amostras microbiológicas foram analisadas de acordo com os parâmetros recomendados pela Resolução n°. 12 de 02/01/2001 da Agência Nacional de Vigilância Sanitária – ANVISA: enumeração de coliformes termotolerantes e pesquisa de Salmonella sp. Além dessas análises, também foram enumeradas a população de bactérias aeróbias mesófilas e de bolores e leveduras e os resultados foram submetidos à anova pelo teste de Tuckey a 5% de probabilidade. Os resultados obtidos nas análises de qualidade microbiológicas demonstram que a beterraba e a cenoura irrigadas com água residuária encontram-se de acordo com os padrões estabelecidos pela legislação vigente (Salmonella sp. ausência em 25 g e coliformes termotolerantes =3 NMP. g-1) e a enumeração de mesófilos e bolores e leveduras não ultrapassaram 4,5x106, contudo se faz necessário estudos mais aprofundados à temática.

X-ray crystallography is one of the most powerful tools for the elucidation of the structure of biological macromolecules such as proteins and viruses. Crystallographic techniques are extensively used for investigations on protein structure, ligand-binding, mechanisms of enzyme catalyzed reactions, protein-protein interactions, role of metal ions in protein structure and function, structure of multi-enzyme complexes and viruses, protein dynamics and for a myriad other problems in structural biology. Crystallographic studies are essential for understanding the intricate details of the mechanism of action of enzymes at molecular level. Understanding the subtle differences between the pathogenic enzymes and host enzymes is necessary for the design of inhibitor molecules that specifically inhibit parasite enzymes. The current thesis deals with the application of biochemical and crystallographic techniques for understanding the structure and function of proteins from two pathogenic organisms – a plant virus Physalis Mottle Virus (PhMV), and a pathogenic bacterium, Salmonella typhimurium and also stress induced proteins from Oryza sativa. The thesis has been divided into seven chapters, with the first four chapters describing the work carried out on PhMV, while the rest of the chapters deal with the studies on stress response proteins from Oryza sativa and Salmonella typhimurium. The first part of the thesis deals with studies on viral capsids. Viruses are obligate parasites that have proteinaceous capsids enclosing the genetic material, which, in the case of small plant viruses, is invariably ss-RNA. X-ray diffraction studies on single crystals of viruses enable visualization of the structures of intact virus particles at near-atomic resolution. These studies provide detailed information regarding the coat protein folding, molecular interactions between protein subunits, flexibility of the N-and C-terminal segments and their probable importance in viral assembly, role of RNA in capsid assembly, nucleic acid (RNA)-protein interactions, the capsid structure and mechanism of assembly and disassembly. The present thesis deals with the capsid structure and analysis of the coat protein (CP) recombinant mutants of PhMV. Virus assembly, one of the important steps in the life cycle of a virus, involves specific interactions between the structural protein and cognate viral genome. This is a complex process that requires precise protein-protein and protein nucleic acid interactions. In fact, most of the biological functional units such as ribosomes and proteosomes also require highly co-ordinated macromolecular interactions for their functional expression. Viruses being simple in their architecture, serve as excellent model systems to understand mechanism of macromolecular assembly and provide necessary information for the development of antiviral therapeutics, especially in animal viruses. PhMV is a plant virus infecting several members of Solanaceae family. It belongs to the tymoviridae group of single stranded RNA viruses. Its genome is encapsidated in a shell comprising of 180 (architecture based on T = 3 icosahedral lattice) chemically identical coat protein (CP) subunits (~ 20,000Da) arranged with icosahedral symmetry. In an earlier phase of work, PhMV purified from infected plant leaves was crystallized in the space group R3 (a = 294.56 Å,  = 59.86). X-ray diffraction data to 3.8 Å resolution were recorded on films by screenless oscillation photography. Using this data of severely limited quality and poor completion (40%), the structure PhMV was determined by molecular replacement using the related turnip yellow mosaic virus (TYMV) structure as the phasing model. There was therefore a need to re-determine and improve the structure, which could be useful for understanding the earlier detailed studies on its biophysical properties. As a continuation of these studies, the present investigations were conceived with the goal of determining the natural top and bottom component capsid structures of PhMV. Investigations were also carried out to examine the possibility of enhancing the diffraction quality of PhMV crystals. The thesis begins with a review of the current literature on the available crystal structures of viruses and their implications for capsid assembly (chapter I). All experimental and computational methods used during the course of investigations are described in chapter II, as most of these are applicable to all the structure determinations and analyses. The experimental procedures described include cloning, overexpression, purification, crystallization and intensity data collection. Computational methods covered include details of various programs used during data processing, structure solution, refinement, model building, validation and analysis. Chapter III describes structural studies on top and bottom components of PhMV. Purified tymoviruses including PhMV are found to contain two classes of particles that sediment at different velocities through sucrose gradients and are called the top (sedimentation coefficient 54 Svedberg units(S)) and the bottom (115S) components. The top component particles are either devoid of RNA or contain only a small subgenomic RNA (5%) while the bottom component particles contain the full length genomic RNA. Only the bottom component is infectious. The top and bottom components were separately crystallized in P1 and R3 space groups, respectively. It is of interest to note that crystals of the bottom component obtained earlier belonged to R3 space group while recombinant capsids that lack of full length RNA as in natural top component crystallized in the P1 space group. A polyalanine model of the homologous TYMV was used as the phasing model to determine the structures of these particles by molecular replacement using the program AMoRe. The refinement of top and bottom component capsid structures were carried out using CNS version 1.1 and the polypeptide models were built into the final electron-density map using the interactive graphics program O. The quality of the map was sufficient for building the model and unambiguous positioning of the side chains. There is a significant difference in the radius of the top and bottom component capsids, the top component being 5 Å larger in radius. Thus, RNA makes the capsid more compact, even though RNA is not a pre-requisite for capsid assembly. Partially ordered RNA was observed in the bottom component. The refined models could form the basis for understanding the architecture, protein-protein interactions, protein-nucleic acid interactions, stability and assembly of PhMV. Chapter IV provides a detailed description of the mutations carried out on PhMV coat protein towards enhancing the diffraction quality of crystals. The gene coding for PhMV coat protein (PhMVCP) and several of its deletion and substitution mutants were originally cloned in pRSETC and pET-21 vectors by Mira Sastri and Uma Shankar in Prof. Savithri’s laboratory at the Department of Biochemistry. It was observed that the recombinant intact coat protein and several mutants lacking up to 30 amino acids from the N-terminal end could assemble into empty shells resembling the natural top component. None of these deletion mutants crystallized in forms that diffracted to high resolution. Based on the intersubunit contacts observed, three more site-specific mutants were designed. These three mutants were expressed in BL21 (DE3), purified and crystallized. Even these mutant crystals did not diffract to high resolution. The polypeptide fold of PhMV coat protein therefore was carefully examined for probable reasons. It was found that PhMV subunit has three major cavities. Three cavities are likely to increase the flexibility of protein subunits, which in turn may result in crystals of poor quality. Mutations V52W, S158Q and A160L were shown to fill up these cavities and with the view of obtaining better crystals. These site specific mutations were carried out the mutant proteins were purified. It was shown that the recombinant capsids are stable and possess T=3 architecture. Two mutants were crystallized and a data set for V52W extending to 6.0 Å resolution could be collected. Due to the limited resolution, further work was not pursued. It is plausible that the triple mutant will diffract to higher resolution. The second part of the thesis deals with stress response proteins from Oryza sativa and Salmonella typhimurium. It is known that viral infection and abiotic and biotic stresses induce a network of proteins in plants. Chapter V presents a review of the current literature on stress proteins, focusing mainly on Oryza sativa and S. typhimurium stress response proteins. Chapter VI describes the over expression of stress proteins SAP1 and SAP2 from rice. These stress related proteins confer tolerance to cold, dehydration and salt stress in rice. These proteins have been cloned in the expression vector pEt-28(a) and expressed in E. coli strain BL21 CodonPlus(DE3)RIL. The proteins were purified and crystallization trials were made. However, there were no hits. In an attempt to get crystals, nine deletion constructs of SAP1 were designed eliminating potentially disordered and unfolded regions based on a bioinformatics analysis. Crystallization trails are being carried out on three of the constructs. Structural studies on a universal stress protein from Salmonella typhimurium, which shares homology with the rice universal stress proteins, was initiated. Apart from this, several other stress related proteins of Salmonella typhimurium have also been selected for structural and functional studies. These include YdaA, YbdQ, Yic, Ynaf, Yec, Spy and Usb. All these were cloned and expressed in E. coli. Out of seven proteins, Ynaf, YdaA and YbdQ were found in the soluble fraction and were expressed in quantities suitable for structural studies. I could crystallize YdaA and Ynaf. X-ray diffraction data to resolutions of 3.6 Å and 2.3 Å were collected on crystals of YdaA and YnaF, respectively. A tentative structure of YnaF has been obtained. Further attempts to determine these structures are in progress. Biophysical, Biochemical functional characterization of YdaA and YnaF proteins are described. Structural studies on mannose-6-phosphate isomerase, an enzyme related to stress regulatory proteins from S. typhimurium are dealt with in Chapter VII. Mannose 6-phosphate isomerase (MPI) catalyzes the interconversion of mannose 6-phosphate and fructose 6-phosphate. The structure could be solved in its apo and holo forms (with two different metal atoms, Y3+ and Zn2+), and complexed with the cyclic form of the substrate fructose 6-phosphate (F6P) and Zn2+. Isomerization involves acid/base catalysis with proton transfer between C1 and C2 atoms of the substrate. Lys 132, His 131, His 99 and Asp 270 are close to the substrate and are likely to be the residues involved in proton transfer. Interactions observed at the active site suggest that the ring opening step is catalyzed by His 99 and Asp 270. An active site loop consisting of residues 130-133 undergoes conformational changes upon substrate binding. The metal ion is not close to the substrate atoms involved in proton transfer. Binding of the metal induces structural order in the loop consisting of residues 50-54. Hence, the metal atom does not appear to play a direct role in catalysis, but is probably important for maintaining the architecture of the active site. Based on these structures and earlier biochemical work, a probable isomerization mechanism has been proposed. The thesis concludes with a brief discussion on the future prospects of the work. The following manuscripts have been published or will be communicated for publication based on the results presented in the thesis:

Total RNA was extracted from chicken oviduct epithelial cells. Avian Beta-defensin (AvBD)-2, -6, and -12 cDNAs were amplified by reverse transcription-PCR and cloned into pRSET A style='msoareast-language:ZH-CN'>, a protein expression vector. The class=SpellE>hexa-histidine-tagged class=SpellE>AvBD peptides were expressed in Escherichia coli (E. coli) BL21(DE3) class=SpellE>plysS and affinity-purified. The antimicrobial activities of the recombinant AvBDs against E. coli style='msoareast-language:ZH-CN;mso-bidiont-style:italic'>, Salmonella class=SpellE>enterica style='mso-bookmark:OLE_LINK9'> serovar Typhimurium (S. class=SpellE>typhimurium), and Staphylococcus aureus style='msoareast-language:ZH-CN'> (S. aureus) were determined. style='msoareast-language:ZH-CN;mso-bidiont-style:italic'> At 8, 16 and 32 µg/ml, all three rAvBDs killed and inhibited the growth style='msoareast-language:ZH-CN'> of E. coli style='msoareast-language:ZH-CN;mso-bidiont-style:italic'>, S. typhimurium, and S. aureus. The killing of rAvBD-2, -6, and -12 against stationary phase E. coli and S. class=SpellE>aureus was pH dependent in the range investigated. style='msoareast-language:ZH-CN'> In addition, the killing-curves showed that rAvBDs exerted their antimicrobial function within 30 minutes of treatment, suggesting the fast killing mechanisms of rAvBDs.

Tableau d’honneur de la Faculté des études supérieures et postdoctorales, 2013-2014. / Les suppléments antibiotiques dans l'alimentation animale sont sévèrement critiqués. Les probiotiques sont une alternative prometteuse, mais la caractérisation de leurs effets demeure nécessaire. Ce mémoire décrit l'influence de Pediococcus acidilactici (PA) et Saccharomyces cerevisiae ssp. boulardii (SCB) sur 1) le microbiote intestinal avant et après le sevrage et, 2) l'immunité et la colonisation intestinale lors d'une infection à Salmonella Typhimurium DT104. Nos résultats montrent que suite au sevrage PA module le microbiote iléal de façon similaire aux antibiotiques tandis que SCB influence le microbiote colique. De plus, SCB seul ou avec PA module certaines populations de cellules immunitaires du sang avant et après l'infection à S. Typhimurium. Cependant, aucun effet n'a été observé sur les autres paramètres évalués. Même si nous approfondissons la compréhension entourant les effets de PA et SCB sur l'hôte, d'autres études sont nécessaires pour optimiser l'utilisation des probiotiques comme alternative aux suppléments d'antibiotiques dans l'élevage. / Antibiotics as growth promoters in pig feed are severely criticized. Probiotics are a promising alternative, but characterization of their effects on the intestinal microbiota and immunity is still necessary. In this study, the influences of Pediococcus acidilactici (PA) and Saccharomyces cerevisiae ssp. boulardii (SCB) on 1) intestinal microbiota before and after weaning and, on 2) immunity and intestinal colonization during a Salmonella Typhimurium DT104 infection were evaluated. Our results show that following weaning PA modulated ileal microbiota similarly to in-feed antibiotic while SCB influenced the colonic microbiota. Moreover, SCB alone or with PA modulate some immune blood cell populations before and after the S. Typhimurium infection. However, no effects were observed on the other parameters assessed. Although we deepened the understanding surrounding the effects of PA and SCB on the host, further studies are needed to fully optimize the use of probiotics as alternatives to antibiotic supplements in animal husbandry.

Made available in DSpace on 2016-06-02T19:02:40Z (GMT). No. of bitstreams: 1 3950.pdf: 2965296 bytes, checksum: 1e00d517bd464f272b8c1a2c9f67ca9c (MD5) Previous issue date: 2011-10-11 / Valée S.A. / Swine erysipelas is among the diseases that causes great economic losses in swine cultures worldwide. The disease is caused by the bacterium Erysipelothrix rhusiopathiae, and the surface protein SpaA is one of its main antigens. Herein, we report studies concerning the development of recombinant vaccines against swine erysipelas based on the SpaA antigen. Protein production for a subunit vaccine formulation was studied in shaken flasks and 5.0 L bioreactors. For this propose, a 1026 bp fragment of the spaA gene was cloned in Escherichia coli cells under the lac promoter control. The recombinant organism (E. coli BL21(DE3) pET28a_spaA) was cultivated in fed batch using complex medium with glycerol as carbon source. Nonconventional induction strategies were evaluated and high protein yield (198 mgprot/gDCW) and productivity values (0.4 gprot/L.h) were reached. The same antigen was cloned for expression and secretion in attenuated Salmonella typhimurium cells to obtain a live bacterial vector for the SpaA antigen. The recombinant lineage was able to express and secrete the SpaA fragment fused to the alpha-hemolysin secretion signal both in vitro and in vivo. High plasmid maintenance was observed in both conditions. The vaccinal vehicle showed to be able to colonize the Peyer patches and to invade the gut epithelial barrier in the inoculated animals. Immunization tests in murine model showed that the recombinant antigen delivered by Salmonella cells inoculated by oral route induced the production of seric IgG antibodies anti-SpaA. According to the literature, these antibodies must be able to promote pathogen opsonization in case of infection, contributing to confer a protective immunity against swine erysipelas to the vaccinated animals. In summary, this work presents contributions to development of subunit vaccines against swine erysipelas, in the form of recombinant protein formulations, or SpaA antigen delivery by attenuated S. typhimurium cells. / A erisipela suína é uma das enfermidades que causam grandes prejuízos na suinocultura em todo o mundo. A doença é causada pela bactéria Erysipelothrix rhusiopathiae, e a proteína de superfície SpaA desse microrganismo é um de seus principais antígenos. Neste trabalho, estudou-se o desenvolvimento de vacinas recombinantes contra a erisipela suína a partir do antígeno SpaA. Avaliou-se a produção de uma vacina de subunidade composta pelo antígeno recombinante, a qual foi estudada em frascos agitados e em biorretores de bancada de 5,0 L. Para isso, um fragmento de 1026 pb do gene spaA foi clonado em células de Escherichia coli sob controle do promotor lac e o organismo recombinante (E. coli BL21(DE3) pET28a_spaA) foi cultivado em batelada alimentada, utilizando-se meio complexo contendo glicerol como fonte de carbono. Estratégias não convencionais de indução foram avaliadas e altos valores de rendimento (198 mgprot/gDCW) e produtividade (0,4 gprot/L.h) da proteína recombinante foram alcançados. O mesmo antígeno foi clonado em um plasmídeo que possibilita a expressão e secreção da proteína recombinante em Salmonella typhimurium atenuada, a fim de se obter um vetor bacteriano vivo para o antígeno em questão. A linhagem recombinante foi capaz de expressar e secretar o fragmento da proteína SpaA fusionado ao sinal de secreção da alfa-hemolisina tanto in vitro quanto in vivo, apresentando alta taxa de manutenção plasmidial nas duas condições. Além disso, o veículo vacinal se mostrou capaz de colonizar as placas de Peyer e de invadir a barreira epitelial do intestino dos animais inoculados. Ensaios de imunização em modelo murino mostraram que a veiculação do antígeno pelas células de Salmonella inoculadas por via oral induziu a produção de anticorpos IgG séricos anti-SpaA, que de acordo com a literatura, devem ser capazes de promover a opsonização do patógeno em caso de infecção, contribuindo para conferir uma imunidade protetora contra a erisipela suína aos animais vacinados. Em suma, este trabalho apresenta contribuições para o desenvolvimento de vacinas de subunidade contra a erisipela suína na forma de uma vacina de proteína recombinante, ou por veiculação do antígeno SpaA por linhagens atenuadas de S. typhimurium.

As infecções por bactérias representam um grave problema hoje e para o futuro, devido ao fato de que estes microrganismos desenvolvem mecanismos de resistência aos antibióticos ao longo do tempo de uso. A formação de biofilmes também é um fator a ser discutido no cenário atual por estar associado a muitas infecções bacterianas humanas, principalmente àquelas envolvendo dispositivos médicos aumentando assim os riscos de infecções hospitalares. O ácido ursólico (AU) é um triterpeno conhecido por suas atividades biológicas relatadas. Apresenta moderada atividade antibacteriana, porém tem demonstrado importante citotoxicidade frente a algumas linhagens celulares. Em vista disso, neste trabalho se desenvolveu uma série de novas moléculas derivadas do AU com alterações nas posições C-3 e C-28. Quatro moléculas com substituição em C-3 (derivados 2, 3, 4e 5) e uma com substituição em C-3 e C-28 (derivado 6) foram comparadas ao AU (1) quanto a atividade antibacteriana. As cepas utilizadas foram Salmonela Typhimurium, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Proteus mirabilis, Staphylococcus epidermidis e Staphylococcus aureus. Os compostos 3 e 6 apresentaram melhor perfil inibitório de forma geral, onde 3 apresentouse bactericida para S. aureus e S. epidermidis (Gram positivas) e paraP. mirabilis (Gram negativa) apresentou-se bacteriostático. / The ursolic acid (UA) is a triterpene known for their biological activities reported. Thus, become useful techniques semi-synthesis starting from natural products extracted, for example residue industries in order to improve the pharmacological properties decreasing toxicity. The bacterial infections are a serious problem today and in the future due to the fact that these organisms develop resistance mechanisms to antibiotics over time of use. The formation of biofilms is also a factor to be discussed in the current scenario because of being responsible for a very high number of rejections and other prosthetic devices by increasing the risk of nosocomial infections. The AU has a moderate antibacterial activity reported in the literature, but showed significant cytotoxicity against some cell lines. In view of this it developed a series of new molecules derived from AU residues extracted from apples (Mallus domestica) from the juice industry by promoting the so-called green chemistry. The molecules undergo changes in C-3 and C-28. Four molecules with substitution at C-3 (derived from 2, 3, 4 and 5) and one with substitution at C-3 and C-28 (derived 6) were compared with au (1). The strains used in the tests of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration were Salmonella Typhimurium, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Proteus mirabilis, Staphylococcus epidermidis and Staphylococcus aureus. Compounds 3 and 6 had better inhibitory profile in general, where three presented bactericidal to S. aureus and S. epidermidis (Gram positive) and P. mirabilis (Gram negative) appeared bacteriostatic.

As infecções por bactérias representam um grave problema hoje e para o futuro, devido ao fato de que estes microrganismos desenvolvem mecanismos de resistência aos antibióticos ao longo do tempo de uso. A formação de biofilmes também é um fator a ser discutido no cenário atual por estar associado a muitas infecções bacterianas humanas, principalmente àquelas envolvendo dispositivos médicos aumentando assim os riscos de infecções hospitalares. O ácido ursólico (AU) é um triterpeno conhecido por suas atividades biológicas relatadas. Apresenta moderada atividade antibacteriana, porém tem demonstrado importante citotoxicidade frente a algumas linhagens celulares. Em vista disso, neste trabalho se desenvolveu uma série de novas moléculas derivadas do AU com alterações nas posições C-3 e C-28. Quatro moléculas com substituição em C-3 (derivados 2, 3, 4e 5) e uma com substituição em C-3 e C-28 (derivado 6) foram comparadas ao AU (1) quanto a atividade antibacteriana. As cepas utilizadas foram Salmonela Typhimurium, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Proteus mirabilis, Staphylococcus epidermidis e Staphylococcus aureus. Os compostos 3 e 6 apresentaram melhor perfil inibitório de forma geral, onde 3 apresentouse bactericida para S. aureus e S. epidermidis (Gram positivas) e paraP. mirabilis (Gram negativa) apresentou-se bacteriostático. / The ursolic acid (UA) is a triterpene known for their biological activities reported. Thus, become useful techniques semi-synthesis starting from natural products extracted, for example residue industries in order to improve the pharmacological properties decreasing toxicity. The bacterial infections are a serious problem today and in the future due to the fact that these organisms develop resistance mechanisms to antibiotics over time of use. The formation of biofilms is also a factor to be discussed in the current scenario because of being responsible for a very high number of rejections and other prosthetic devices by increasing the risk of nosocomial infections. The AU has a moderate antibacterial activity reported in the literature, but showed significant cytotoxicity against some cell lines. In view of this it developed a series of new molecules derived from AU residues extracted from apples (Mallus domestica) from the juice industry by promoting the so-called green chemistry. The molecules undergo changes in C-3 and C-28. Four molecules with substitution at C-3 (derived from 2, 3, 4 and 5) and one with substitution at C-3 and C-28 (derived 6) were compared with au (1). The strains used in the tests of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration were Salmonella Typhimurium, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Proteus mirabilis, Staphylococcus epidermidis and Staphylococcus aureus. Compounds 3 and 6 had better inhibitory profile in general, where three presented bactericidal to S. aureus and S. epidermidis (Gram positive) and P. mirabilis (Gram negative) appeared bacteriostatic.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A mutagenicidade do material particulado é atribuída primeiramente aos hidrocarbonetos policíclicos aromáticos (HPA). Investigamos a atividade mutagênica do material particulado (MP2,5) em amostras coletadas em três pontos da cidade do Rio de Janeiro. As coletas foram realizadas com auxílio de um amostrador de grande volume na Avenida Brasil, no campus da Universidade do Estado do Rio de Janeiro e no Túnel Rebouças em filtros de fibra de vidro. Metade de cada filtro foi submetido à extração por sonicação com o solvente diclorometano. Seis HPA foram identificados e quantificados por cromatografia gasosa com espectrometria de massa (GC/MS). Após a análise química as concentrações dos HPA obtidos foram correlacionados ao fatores físicos, além de ser realizado avaliação de risco para cada HPA estudado. Linhagens de Salmonella typhimurium (TA98 e derivadas TA98/1.8-DNP6, YG1021 e YG1024) foram utilizadas no ensaio de mutagenicidade e tratadas (10-50 g/placa) com extrato orgânico na presença e na ausência de metabolização exógena. Células de raiz de cebola foram tratadas com extratos orgânicos nas concentrações (5-25g/mL). A alta umidade encontrada no Túnel Rebouças pode ter influenciado na deposição de cinco dos seis HPA estudados em material particulado. Além disso, em diferentes condições de tráfego, motoristas de ônibus que cruzam a Avenida Brasil e o Rebouças túnel estão expostos ao risco induzidos por HPA na ordem de 10-6. Mutagenicidade foi detectada tanto na presença quanto na ausência de metabolização, para as linhagens YG1021 e YG1024 nos três pontos, sugerindo a presença de nitro e amino derivados de HPA. As amostras do Túnel Rebouças apresentaram os maiores valores para rev/g e rev/m3. Estes resultados podem estar relacionados ao longo trajeto e a restrita ventilação. Efeito citotóxico foi detectado pelo ensaio Allium cepa nos três pontos de monitoramento. Além disso os extratos orgânicos provenientes das coletas da Avenida Brasil, UERJ e do Túnel Rebouças induziram efeito clastogênico em células de raiz de Allium cepa / The mutagenicity of airborne particles from combustion is attributed primarily to polycyclic aromatic hydrocarbons (PAHs). We investigated the mutagenic activity of particulate matter (PM2.5) samples collected from three sites in Rio de Janeiro. Samples were collected using a high-volume sampler at Avenida Brasil, the campus of the Rio de Janeiro State University, and Rebouças tunnel. Half of each filter was submitted to sequential extraction by sonication with dichloromethane. Six PAHs were quantified by gas chromatography/mass spectrometry (GC/MS). Salmonella typhimurium TA98 and the derivative strains TA98/1.8-DNP6, YG1021 and YG1024 used in mutagenicity assays were treated (10-50 g/plate) with and without exogenous metabolization. Onion root cells were treated with organic extracts concentrations (5-25g/mL). The high humidity detected in the Rebouças Tunnel may have influenced the deposition of five of the six PAHs studied in particulate matter. Moreover, in different traffic conditions can put the bus drivers that cross the Brasil Avenue and Rebouças tunnel at the risk of exposure induced by HPA in the order of 10-6. Independently of exogenous metabolization, mutagenicity was detected for strains YG1021 and YG1024 at all the sites, suggesting the presence of nitro and amino derivatives of PAHs. Rebouças tunnel presented the highest values for rev/g and rev/m3. These could be related to the fact that this long, enclosed passageway and restricts ventilation. Cytotoxic effect was detected by the Allium cepa test in the three monitoring sites. Also organic extracts from Brazil Avenue, UERJ and Rebouças Tunnel showed clastogenic effect in Allium cepa roots

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A mutagenicidade do material particulado é atribuída primeiramente aos hidrocarbonetos policíclicos aromáticos (HPA). Investigamos a atividade mutagênica do material particulado (MP2,5) em amostras coletadas em três pontos da cidade do Rio de Janeiro. As coletas foram realizadas com auxílio de um amostrador de grande volume na Avenida Brasil, no campus da Universidade do Estado do Rio de Janeiro e no Túnel Rebouças em filtros de fibra de vidro. Metade de cada filtro foi submetido à extração por sonicação com o solvente diclorometano. Seis HPA foram identificados e quantificados por cromatografia gasosa com espectrometria de massa (GC/MS). Após a análise química as concentrações dos HPA obtidos foram correlacionados ao fatores físicos, além de ser realizado avaliação de risco para cada HPA estudado. Linhagens de Salmonella typhimurium (TA98 e derivadas TA98/1.8-DNP6, YG1021 e YG1024) foram utilizadas no ensaio de mutagenicidade e tratadas (10-50 g/placa) com extrato orgânico na presença e na ausência de metabolização exógena. Células de raiz de cebola foram tratadas com extratos orgânicos nas concentrações (5-25g/mL). A alta umidade encontrada no Túnel Rebouças pode ter influenciado na deposição de cinco dos seis HPA estudados em material particulado. Além disso, em diferentes condições de tráfego, motoristas de ônibus que cruzam a Avenida Brasil e o Rebouças túnel estão expostos ao risco induzidos por HPA na ordem de 10-6. Mutagenicidade foi detectada tanto na presença quanto na ausência de metabolização, para as linhagens YG1021 e YG1024 nos três pontos, sugerindo a presença de nitro e amino derivados de HPA. As amostras do Túnel Rebouças apresentaram os maiores valores para rev/g e rev/m3. Estes resultados podem estar relacionados ao longo trajeto e a restrita ventilação. Efeito citotóxico foi detectado pelo ensaio Allium cepa nos três pontos de monitoramento. Além disso os extratos orgânicos provenientes das coletas da Avenida Brasil, UERJ e do Túnel Rebouças induziram efeito clastogênico em células de raiz de Allium cepa / The mutagenicity of airborne particles from combustion is attributed primarily to polycyclic aromatic hydrocarbons (PAHs). We investigated the mutagenic activity of particulate matter (PM2.5) samples collected from three sites in Rio de Janeiro. Samples were collected using a high-volume sampler at Avenida Brasil, the campus of the Rio de Janeiro State University, and Rebouças tunnel. Half of each filter was submitted to sequential extraction by sonication with dichloromethane. Six PAHs were quantified by gas chromatography/mass spectrometry (GC/MS). Salmonella typhimurium TA98 and the derivative strains TA98/1.8-DNP6, YG1021 and YG1024 used in mutagenicity assays were treated (10-50 g/plate) with and without exogenous metabolization. Onion root cells were treated with organic extracts concentrations (5-25g/mL). The high humidity detected in the Rebouças Tunnel may have influenced the deposition of five of the six PAHs studied in particulate matter. Moreover, in different traffic conditions can put the bus drivers that cross the Brasil Avenue and Rebouças tunnel at the risk of exposure induced by HPA in the order of 10-6. Independently of exogenous metabolization, mutagenicity was detected for strains YG1021 and YG1024 at all the sites, suggesting the presence of nitro and amino derivatives of PAHs. Rebouças tunnel presented the highest values for rev/g and rev/m3. These could be related to the fact that this long, enclosed passageway and restricts ventilation. Cytotoxic effect was detected by the Allium cepa test in the three monitoring sites. Also organic extracts from Brazil Avenue, UERJ and Rebouças Tunnel showed clastogenic effect in Allium cepa roots

As infecções por bactérias representam um grave problema hoje e para o futuro, devido ao fato de que estes microrganismos desenvolvem mecanismos de resistência aos antibióticos ao longo do tempo de uso. A formação de biofilmes também é um fator a ser discutido no cenário atual por estar associado a muitas infecções bacterianas humanas, principalmente àquelas envolvendo dispositivos médicos aumentando assim os riscos de infecções hospitalares. O ácido ursólico (AU) é um triterpeno conhecido por suas atividades biológicas relatadas. Apresenta moderada atividade antibacteriana, porém tem demonstrado importante citotoxicidade frente a algumas linhagens celulares. Em vista disso, neste trabalho se desenvolveu uma série de novas moléculas derivadas do AU com alterações nas posições C-3 e C-28. Quatro moléculas com substituição em C-3 (derivados 2, 3, 4e 5) e uma com substituição em C-3 e C-28 (derivado 6) foram comparadas ao AU (1) quanto a atividade antibacteriana. As cepas utilizadas foram Salmonela Typhimurium, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Proteus mirabilis, Staphylococcus epidermidis e Staphylococcus aureus. Os compostos 3 e 6 apresentaram melhor perfil inibitório de forma geral, onde 3 apresentouse bactericida para S. aureus e S. epidermidis (Gram positivas) e paraP. mirabilis (Gram negativa) apresentou-se bacteriostático. / The ursolic acid (UA) is a triterpene known for their biological activities reported. Thus, become useful techniques semi-synthesis starting from natural products extracted, for example residue industries in order to improve the pharmacological properties decreasing toxicity. The bacterial infections are a serious problem today and in the future due to the fact that these organisms develop resistance mechanisms to antibiotics over time of use. The formation of biofilms is also a factor to be discussed in the current scenario because of being responsible for a very high number of rejections and other prosthetic devices by increasing the risk of nosocomial infections. The AU has a moderate antibacterial activity reported in the literature, but showed significant cytotoxicity against some cell lines. In view of this it developed a series of new molecules derived from AU residues extracted from apples (Mallus domestica) from the juice industry by promoting the so-called green chemistry. The molecules undergo changes in C-3 and C-28. Four molecules with substitution at C-3 (derived from 2, 3, 4 and 5) and one with substitution at C-3 and C-28 (derived 6) were compared with au (1). The strains used in the tests of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration were Salmonella Typhimurium, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Proteus mirabilis, Staphylococcus epidermidis and Staphylococcus aureus. Compounds 3 and 6 had better inhibitory profile in general, where three presented bactericidal to S. aureus and S. epidermidis (Gram positive) and P. mirabilis (Gram negative) appeared bacteriostatic.