<b>Background</b> Antibiotic resistance is an important societal health issue. The greatest risk factor for developing a resistant infection is antibiotic use. Almost 75% of all antibiotics in the UK are prescribed in the community, and mostly for acute respiratory tract infections (RTIs). Yet, the majority of RTIs are self-limiting, viral and do not need antibiotic treatment, especially in young children. While the effects of antibiotic-resistant infections have been widely studied in hospitals (e.g. the MRSA 'superbug'), we know less about how antibiotic-resistant infections affect people in the community, even though this is where most antibiotics are prescribed. There is also widespread public misconception about antibiotic use and resistance despite several high-profile, multimillion antibiotic awareness campaigns. This is important to address because consultation behaviour and expectations for antibiotics are a significant determinant of antibiotic use in the community. <b>Methods</b> Three studies were conducted for this thesis. First, a systematic review and meta-analysis to assess the evidence of the impact of antibiotic resistance for patients with common infections in the community; second, a retrospective analysis of routinely collected primary care data to examine the relationship between antibiotic exposure and antibiotic 'response failure' in preschool children presenting with acute RTIs; third, a qualitative interview study to explore parents' perceptions and understanding of antibiotic use and resistance when they consider consulting in the community with their preschool child who has a respiratory tract infection. <b>Results</b> Antibiotic resistance significantly impacts on patients' illness burden for common infections in the community. Patients who presented in community health care settings with antibiotic-resistant E. coli urinary tract infections and S. pneumoniae respiratory tract infections were more likely to experience delays in recovery after antibiotic treatment. From routinely collected primary data (2009-2016), preschool children receiving two or more antibiotic courses in the previous year for acute RTIs had greater likelihood of antibiotic 'response failure' to treatment for subsequent acute RTIs compared to children that had received no previous antibiotics. When interviewing parents of young children, most parents were quite reticent about antibiotics for their children. However, very few considered antibiotic resistance as a possible harm of antibiotics. Parents thought their families were at low risk of antibiotic resistance because their families were 'low users' of antibiotics and did not perceive any association between their individual consumption of antibiotics and the development and spread of antibiotic-resistant bacteria in the community. They wanted future antibiotic awareness campaigns to have a universal message relevant to their families that fit into their daily lives. <b>Conclusions</b> The findings challenge the perception that antibiotic prescribing and resistance in the community poses little or no additional risk to patients in the community, or is remote from everyday prescribing decisions. Rapid diagnostic tests and other prognostic tools need to be promoted and evaluated to better identify patients who might need an antibiotic, and reduce the risk antibiotic response failures. Clinicians and parents should exercise caution about whether further antibiotics for acute RTIs are likely to be beneficial in those children who have received two or more antibiotic prescriptions for acute RTIs during the previous 12 months. Incorporating this into clinical practice guidelines and decision-support systems will help clinicians and parents consider a non-antibiotic strategy for acute RTIs. Future guidelines, campaigns and interventions around antibiotic resistance should tailor initiatives to outcomes that patients and clinicians in the community can relate to and slot into their daily lives. More research is needed to evaluate the impact of other common infections in primary care, and determine the relative contribution of antibiotic resistance to patients not responding to antibiotic treatment for common infections.

This diploma thesis deals with resistance of microorganisms to antibiotics and public awareness of this topic. The aim of the thesis was to find out what the public awareness of the given topic is and what knowledge the public has about the possibilities of prevention of creation and spreading of resistant microorganisms. Three main hypotheses have been set: Hypothesis 1: The public has no deeper knowledge of antibiotic treatment. Hypothesis 2: The public will in most cases get information on antibiotic treatment from a doctor. Hypothesis 3: Most people are unaware that they can contribute by rational use of antibiotics to prevent antibiotic resistance. In order to achieve the stated goals of the diploma thesis, a quantitative method of research was used. Data collection was conducted through anonymous questionnaire survey of the public population using the Click4Survey.cz web server and distribution of paper questionnaires in FN Plzeň. Results of the questionnaire survey were evaluated based on statistical data processing in Microsoft Office Excel 2007, and a Chi-squared test of independence in the pivot table and a relative frequency test were used to verify the hypotheses. Based on the evaluation of the results, it was found that among the groups of respondents, depending on whether they filled in a questionnaire on the Internet or a printed questionnaire at the hospital, there are fundamental statistical differences in knowledge and awareness of the problem. That is why it was necessary to further evaluate these two groups separately. Antibiotic resistance nowadays is a very important medical issue. The main cause is inconsistent use or overuse of antibiotics in both human and veterinary medicine, as well as insufficient awareness of the extent of the problem and the consequences of antibiotic resistance. For this reason, I have chosen this issue as a topic for my diploma thesis to highlight the risks of antibiotic resistance and to provide information primarily to the general public.

Submitted by ANDRÉ LUIZ NAGATANI RIGUEIRO null (andrenagatani@yahoo.com.br) on 2016-03-21T15:13:20Z No. of bitstreams: 1 Defesa-André04JAN2016_FINAL.pdf: 1050041 bytes, checksum: ad145a930ac44964a3ccf42de7b60ee7 (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-03-23T12:00:02Z (GMT) No. of bitstreams: 1 rigueiro_aln_me_dra.pdf: 1050041 bytes, checksum: ad145a930ac44964a3ccf42de7b60ee7 (MD5) / Made available in DSpace on 2016-03-23T12:00:02Z (GMT). No. of bitstreams: 1 rigueiro_aln_me_dra.pdf: 1050041 bytes, checksum: ad145a930ac44964a3ccf42de7b60ee7 (MD5) Previous issue date: 2016-01-14 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O objetivo deste estudo foi avaliar o efeito do uso combinado de monensina sódica (MON) e virginiamicina (VM) sobre o desempenho produtivo, comportamento ingestivo, características de carcaça, e saúde ruminal de bovinos Nelore terminados em confinamento. Foram utilizados 72 animais machos inteiros da raça Nelore, com peso vivo médio inicial de 388±31,07 kg, provindos de sistema de recria em pasto. Os animais foram divididos em quatro tratamentos de acordo com associação ou não dos aditivos nas fases de adaptação e terminação: MON (30 mg/kg) na adaptação + terminação; MON (30 mg/kg) + VM (25 mg/kg) na adaptação e VM (25 mg/kg) na terminação; MON (30 mg/kg) + VM (25 mg/kg) na adaptação e VM (25 mg/kg) + MON (30 mg/kg) na terminação e VM (25 mg/kg) na adaptação e MON (30 mg/kg) + VM (25 mg/kg) na terminação, caracterizando assim delineamento em blocos casualizados, em que cada tratamento contou com 6 repetições (3 animais por baia, sendo as baias as unidades experimentais). A duração do experimento foi de 90 dias, sendo 19 dias de adaptação, divididos em três períodos (7, 5 e 7 dias) com dietas de 69%, 74% e 79% de ingredientes concentrados, respectivamente; e 71 dias de terminação, com dieta contendo 84% de concentrado. Durante a fase de adaptação, a suplementação somente com VM aumentou a ingestão de matéria seca em quilos (IMSKG), ingestão de matéria seca em % do peso vivo (IMSPV), diminuiu a flutuação da ingestão de matéria seca (IMS) em quilos e em porcentagem e diminui os dias para os animais atingirem 2% de IMSPV (P<0,05). No período total de confinamento, a suplementação com MON e VM na fase de adaptação e somente VM na terminação aumentou o PV final, IMSKG, IMSPV, GPD, peso de carcaça quente, área de olho de lombo (AOL) final e ganho de AOL por dia. Não houve diferenças significativas entre os tratamentos para os parâmetros relacionados a saúde ruminal e seletividade da ração (P>0,05). Desta forma, a suplementação com somente VM melhorou o desempenho dos animais na fase de adaptação, no entanto, a suplementação com MON e VM na fase de adaptação e somente VM na terminação melhorou o desempenho e características de carcaça dos animais sem diferenças nos parâmetros relacionados a saúde ruminal e seletividade da dieta. / The experiment was designed to evaluate the effect of combined use of monensin (MON) and virginiamycin (VM) in the adaptation period and finishing period on feedlot performance, feeding behavior, ruminal health and carcass characteristics of Nellore cattle. Seventy two 26-mo-old yearling Nellore bulls (388±31.07 kg), backgrounded on pasture were randomly allocated and kept in 24 pens (10 m² and e 1.67 m bunk space per animal) according to the treatments: MON (30 mg/kg) in the adaptation and finishing period, MON (30 mg/kg ) + VM (25 mg/kg) in adaptation and only VM (25 mg/kg) in finish, MON (30 mg/kg) + VM (25 mg / kg) in adaptation and VM (25 mg/kg) + MON (30 mg/kg) in finish and VM (25 mg/kg) in adaptation MON (30 mg/kg) + VM (25 mg/kg) in finish, thus characterizing a completely randomized block design. Each treatment was composed by 6 replications (pens) in this study. The adaptation program consisted of ad libitum feeding of three adaptation diets over period of 19 days with concentrate level increasing from 69% to 84% of diet dry matter. During the adaptation period, animals feed with only VM showed greater dry matter intake in kilos, dry matter intake expressed as % of body weight, reduced in dry matter intake variation in kilos and percentage, and the number of days to achieve 2% of DMI, expressed as % of body weight (P<0.05). Throughout the study, steers that were fed MON and VM during the adaptation period and only VM during the finishing period increase final BW, DMI in kilograms and as percentage of BW, average daily gain, hot carcass weight, ribeye area and daily gain of ribeye area when compared to animals receiving others combinations. There were no differences between the treatments for parameters related of rumen health and selective consumption of ration (P>0.05). Thus, supplementation with only VM in the adaptation period improved the feedlot performance of the animals during the adaptation period; however, the supplementation with MON e VM during the adaptation period and only VM in the finishing period improved animals performance and carcass traits of Nellore cattle. / FAPESP: 2013/21672-7

Introdução: Sepse relacionada ao cateter é a principal causa de hospitalizações e a segunda causa de morte em pacientes com insuficiência renal crônica em hemodiálise. O uso de medicações que possam diminuir a incidência de bacteremia relacionada ao cateter parece uma alternativa para diminuir o número de eventos. Objetivo: Comparação do uso de heparina contra minociclina e edetato dissódico (MEDTA) como selo de cateter de longa permanência em pacientes com insuficiência renal crônica em hemodiálise, comparando desfecho de sobrevida de cateter em relação ao tempo livre de bacteremias, alterações de fluxo, necessidade de trombolítico e cultura positiva de material de descarte. Método: Ensaio clínico, randomizado, multicêntrico, aberto, controlado, comparativo entre heparina e MEDTA. Resultados: 28 pacientes foram incluídos no total do estudo (14 no grupo heparina e 14 no grupo MEDTA). Os grupos não apresentaram diferenças estatisticamente significativas em relação à idade, sexo, níveis de albumina e hemoglobina, tempo de permanência de cateter, alterações de fluxo do cateter (fluxo < 250mL/min) e necessidade de uso de trombolítico. O grupo MEDTA apresentou significativamente menos episódios de bacteremia e menor número de culturais de material de descarte positivos (P< 0,05). Conclusões: O uso de MEDTA como selo parece diminuir a incidência de bacteremias em pacientes com insuficiência renal crônica em hemdiálise emuso de acesso vascular por cateter, apresentando índices semelhantes de patência de cateter. / Introduction: Catheter-related sepsis is the leading cause of hospitalization and the second cause of death in patients with chronic renal failure undergoing hemodialysis. The use of medications that can reduce the incidence of catheter-related bacteremia seems as an alternative to reduce the number of events. Objective: Comparison of heparin against minocycline and disodium edetate (MEDTA) as a tunneled catheter lock in patients with chronic renal failure on hemodialysis, comparing outcome of catheter survival over time free survival time of bacteremia, flow alterations, need of thrombolytic therapy and positive culture of material from discard. Method: Clinical trial, randomized, multicenter, open, controlled comparative between heparin and MEDTA. Results: 28 patients were included in the overall study (14 in the heparin group and 14 in MEDTA group). The groups showed no statistically significant differences regarding age, sex, albumin and hemoglobin levels, length of catheter, catheter flow changes (flow <250mL/min) and required use of thrombolytic therapy. MEDTA group had significantly fewer episodes of bacteremia and fewer positive cultural of discarded material (P <0.05). Conclusions: The use of MEDTA as a catheter lock appears to decrease the incidence of bacteremia in patients with chronic renal failure in hemdiálise inuse vascular access catheter, showing similar rates of patency of the catheter.

L'étape finale de biosynthèse du peptidoglycane est catalysée par les D,D-transpeptidases (PBPs), l'une des cibles principales des antibiotiques de type beta-lactame. Récemment, il a été montré qu'une nouvelle classe d'enzymes, les L,D-transpeptidases (LDts), permet de contourner l'inhibition des PBPs. Ces LDts ont été identifiées tant dans des bactéries résistantes aux beta-lactames que dans des formes dormantes de Mycobacterium tuberculosis. Les seuls beta-lactames capables de les inhiber, les carbapénèmes, forment une liaison covalente avec la cystéine catalytique des LDts. Ni le mécanisme de cette inactivation, ni la spécificité de ces enzymes pour les carbapénèmes ne sont toutefois expliqués à ce jour. Le but du présent travail consiste en l'investigation par RMN du mécanisme d'acylation des LDts par ces antibiotiques. Dans ce contexte, la première partie de cette thèse s'intéresse à la compréhension actuelle de l'émergence de ce phénomène de résistance. La seconde partie traite des principes de la RMN et des implémentations développées pour étudier la structure, la thermodynamique et la dynamique des LDts. La troisième et dernière partie démontre le succès de l'approche RMN dans l'étude des diverses étapes de la réaction d'acylation, à travers une étude détaillée de l'apoenzyme, de complexes non covalents avec différents beta-lactames, et de l'enzyme acylée par un carbapénème. Au cours de cette étude, la structure du site actif de l'apoenzyme de Bacillus subtilis a été affinée par rapport à une étude cristallographique antérieure. Pour cette enzyme et son pendant chez Enterococcus faecium, nous avons démontré que la spécificité pour les carbapénèmes n'intervient pas au stade de la formation du complexe non covalent. Pour finir, la formation de la liaison covalente entre LDt et carbapénème induit un réarrangement conformationnel substantiel et une augmentation de la flexibilité de l'enzyme. / The final cross-linking step of the peptidoglycan synthesis is usually catalyzed by D,D-transpeptidases (PBPs), one of the main targets of beta-lactam antibiotics. Recently, it was shown that these PBPs can be by-passed by a novel class of enzymes, the L,D-transpeptidases (LDts), identified in beta-lactam-resistant bacteria as well as in dormant forms of Mycobacterium tuberculosis. The only beta-lactams enable to inactivate these enzymes belong to the carbapenem class. The beta-lactam ring of this antibiotic family then covalently binds the catalytic cysteine of the LDt. Neither the mechanism of this reaction nor the specificity for carbapenems are yet understood. The aim of the present work is to investigate the acylation mechanism of LDts with carbapenems by NMR. In this context, the first part of this thesis focuses on the current biological understanding of the emergence of this resistance pathway. The second part deals with the NMR principles and the implementations developed to study the structure, thermodynamics and dynamics of LDts. The third part demonstrates that NMR is successful in studying all the steps of the acylation reaction. For this purpose, the LDt apoenzyme, the non-covalent complex with various beta-lactams, and the LDt-carbapenem acylenzyme were thoroughly investigated. The structure of the active site of the Bacillus subtilis apoenzyme was refined with respect to a previous crystallographic study. For the latter and the Enterococcus faecium enzymes, we showed that the carbapenem specificity does not occur at the stage of the non-covalent binding. In contrast to non-covalent interactions, the formation of the covalent bond between LDts and carbapenems induces substantial conformational rearrangement and increased flexibility in the enzyme.

Resumo: Dentre os agentes bacterianos amplamente distribuídos no ecossistema aquático, destacam-se as famílias Aeromonadaceae e Enterobactereaceae. Os peixes são importantes veículos de infecções humanas causadas por essas bactérias. Com este enfoque, o estudo foi realizado com o objetivo de verificar a ocorrência de Aeromonas sp., coliformes termotolerantes e bactérias heterotróficas mesófilas em pisciculturas da Região da Baixada Ocidental Maranhense. Para tal, foram selecionadas no período de outubro de 2008 a março de 2009, doze propriedades nos municípios de Pinheiro, Palmeirândia e Perimirim e colhidas amostras de água dos viveiros e peixes de cada piscicultura, totalizando 96 amostras. Em 100% das amostras analisadas foi confirmada a presença de Aeromonas sp., classificadas em quatro espécies, A. hydrophila (87,03%), A. caviae (8,02%), A. veronii sobria (3,70%), A. schubertii (1,23%). Essas ainda apresentaram elevados percentuais de resistência e multiresistência a 12 antimicrobianos testados. As populações de bactérias heterotróficas mesófilas nas pisciculturas variaram de 102 UFC/mL a 104 UFC/mL de água. Das pisciculturas avaliadas, sete apresentaram pelo menos uma amostra em desacordo com o padrão para coliformes termotolerantes. As amostras analisadas revelaram - se como possíveis vias de transmissão de aeromonas potencialmente patogênicas para peixes e ser humano, representando risco para a saúde da população consumidora dos organismos cultivados nessas propriedades / Abstract: Among widely distributed agents in the aquatic ecosystem can be outstanding the families Aeromonadaceae and Enterobacteriaceae. The fishs are very important vehicles of human infections caused for these bacteria. With the approach the study intended to verify the occurrence of Aeromonas sp., thermotolerant coliforms and heterotrophic mesophilic bacteria in fish farms located in Occidental Baixada Maranhense Region. Twelve properties in the Pinheiro, Palmeirândia and Perimirim' s cities were selected in the period from October of 2008 to March of 2009, and harvested water pond and fish samples of each fish farm, with the total of 96 samples. Aeromonas sp. was confirmed in 100% of samples, classified in four species, A. hydrophila (87,03%), A. caviae (8,02%), A. veronii sobria (3,70%), A.schubertii (1,23%). These bacteria showed high resistance and multiple resistance to the 12 antibiotics tested. The populations of heterotrophic mesophilic bacteria varied between 1,4 x 102 UFC/mL to 7,2 x 103 UFC/mL/. Seven fish farms showed at least one sample in disagreement with the standard to termotholerant coliforms. The samples of water and fish revealed the possible sources of potentially pathogenic contamination of aeromonas for fish and human being representing risk for health of the population healthy that consume the organisms cultivated in these properties / Orientador: Oswaldo Durival Rossi Junior / Coorientadora: Francisca Neide Costa / Banca: Henrique Cesar Pereira Figueiredo / Banca: Naiá Carla Marchi de Rezende Lago / Banca: Fabiana Pilarski / Banca: Luiz Augusto do Amaral / Doutor

Orientadora: Eliana Gertrudes Macedo Lemos / Banca: Lúcia Maria Carareto Alves / Banca: Vanderlei Rodrigues / Resumo: Metabólitos secundários são compostos bioativos, com grande importância para a indústria farmacêutica e agropecuária, produzidos por certos grupos de microrganismos e plantas. Os policetídeos, que são sintetizados por complexos enzimáticos denominados policetídeos sintases (PKSs), desatacam-se entre os metabólitos secundários conhecidos e compõe a estrutura química básica de vários antibióticos. Todos os genes envolvidos na biossíntese de um policetídeo se encontram agrupados fisicamente no cromossomo, e contém genes que são altamente conservados, comumente chamados d~ pks mínima. Os métodos tradicionais para pesquisa de novas drogas, que envolvem o cultivo de microrganismos isolados do solo, não são mais tão promissores, devido à alta taxa de redescoberta de antibióticos já conhecidos, que chega a 99,9%, e à pequena parcela de microrganismos do solo que são cultiváveis pelas técnicas padrões de cultivo, cerca de 1 %. A Metagenômica é uma abordagem promissora que permite acessar o genoma desses organismos incultiváveis, pois consiste na extração de DNA diretamente do ambiente e construção de uma biblioteca com este genoma misto. Neste trabalho descrevemos a construção de uma biblioteca feita com DNA de alto peso molecular isolado diretamente de solo coletado sob arboreto de eucaliptos no Estado de São Paulo, Brasil. A biblioteca possui 9.320 clones e foi construída em vetor cosmídeo, com insertos de tamanho variando entre 30 e 45kb...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Secondary metabolites are bioactive compounds with great importance in the pharmaceutical and agriculture industries, procuced by a few groups of microrganisms and plants. The polyketides that are synthetized by enzimatic complexes, denominated polyketides synthases, outstand among the secondary known metabolites, which are part of the main structure of many antibiotics. Ali genes involved in the biosynthesis of antibiotics are found as clusters in the chromossome. The traditional methods for the research of new drugs that are made from microrganisms cultures isolated from the soil are not so promissing, due to the high rate of rediscorevy of already known species, reaching 99.9%. The other small piece of microrganisms are culturable by standards culture methods, reaching 1 % maximum. Metagenomics is a promissing approach that allows the access to genom of these organisms that are not culturable, as it is carried out by DNA extraction directly from the environment and construction of a mixed genomic library. In this work, we describe the construction of a library made from high molecular weight DNA isolated directly form the soi! undemeath a pinus forest in the State of São Paulo, Brazil. The library shows 9.320 dones and it was constructed in a cosmideo vector, with insert size ranging from 30 to 45 kb. Digestion with difterent restriction enzymes of cosmidial DNA randomly chosen allowed to visualize evident difterences in the restriction fragments among the clones, as does the possibility to determine the average insert size. The initial evaluation of the presence of genes involved in the biosynthesis of antibiotics synthesized by the enzymatic system PKS of kind I, was accomplished by the PCR amplification of clones from the library using specific primers. We studied 4.320 clones and the results suggest a great variety of these genes. The PCR products obtained were sequenced for the determination of identity of the amplified gene. / Mestre

O uso de antibióticos para reduzir a infecção pós-operatória em cirurgia de terceiro molar permanece controverso. O objetivo deste estudo foi avaliar a eficácia da terapêutica com Amoxicilina em múltiplas doses para a prevenção de infecção em pacientes submetidos à extração de terceiro molar inferior com posição 2B (classificação de Pell & Gregory). O estudo foi prospectivo, cego, randomizado, duplo, controlado por placebo, com 32 pacientes. Cada paciente atuou como seu próprio controle usando a técnica de boca dividida. Um terceiro molar inferior foi retirado sob a cobertura de antibióticos no pós-operatório (500 mg de amoxicilina a cada 8 horas durante 7 dias) e o outro foi removido sem cobertura antibiótica pós-operatória (cápsulas de placebo a cada 8 horas durante 7 dias), mas em ambas cirurgias foi administrada dose única no pré-operatório de 1 g de amoxicilina. Os seguintes parâmetros foram avaliados no pré-operatório, no quarto e sétimo dias pós-operatórios: trismo, edema facial, temperatura corporal, linfadenopatia, infecção, disfagia e dor. Não houve diferença estatisticamente significativa nos parâmetros avaliados entre os pacientes operados (p> 0,05). Resultado do estudo mostrou que a administração de amoxicilina em dose única pré-operatória e em doses múltiplas pós-operatórias não se mostrou mais eficaz do que a administração somente em dose única pré-operatória com relação aos parâmetros clínicos avaliados nas exodontias de terceiros molares inferiores. / The use of antibiotics to reduce postoperative infection in third molar surgery remains controversial. The goal of this study was to evaluate the efficacy of multi-dose Amoxicilina therapy for the prevention of infection in patients undergoing lower third molar extraction with position 2B (Pell & Gregory classification). The study was a prospective, randomized, double blind, placebo-controlled trial with 32 patients. Each patient acted as their own control using the split-mouth technique. One lower third molar were removed under antibiotic cover postoperative (500 mg amoxicillin 8 hourly for 7 days) and the other were removed without antibiotic cover postoperative (placebo capsules 8 hourly for 7 days), but both surgery was administered preoperatively single dose of 1g of amoxicillin. The following parameters were evaluated on the preoperative and fourth, seventh days postoperative: trismus, facial swelling, body temperature, Lymphadenopathy, infection, dysphagia and pain. There was no statistically significant difference in the parameters evaluated between patients operated (p > 0.05). Results of the study showed that administration of amoxicillin single dose preoperative and postoperative multiple doses was not more effective than single dose administration only pre-operative with respect to clinical parameters evaluated in the lower third molar extractions.

Made available in DSpace on 2014-06-11T19:27:23Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-02-28Bitstream added on 2014-06-13T20:47:49Z : No. of bitstreams: 1 schuch_v_me_jabo.pdf: 3089029 bytes, checksum: 0835ef08e49e97cfdf7ad571bdfc3671 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Metabólitos secundários são compostos bioativos, com grande importância para a indústria farmacêutica e agropecuária, produzidos por certos grupos de microrganismos e plantas. Os policetídeos, que são sintetizados por complexos enzimáticos denominados policetídeos sintases (PKSs), desatacam-se entre os metabólitos secundários conhecidos e compõe a estrutura química básica de vários antibióticos. Todos os genes envolvidos na biossíntese de um policetídeo se encontram agrupados fisicamente no cromossomo, e contém genes que são altamente conservados, comumente chamados d~ pks mínima. Os métodos tradicionais para pesquisa de novas drogas, que envolvem o cultivo de microrganismos isolados do solo, não são mais tão promissores, devido à alta taxa de redescoberta de antibióticos já conhecidos, que chega a 99,9%, e à pequena parcela de microrganismos do solo que são cultiváveis pelas técnicas padrões de cultivo, cerca de 1 %. A Metagenômica é uma abordagem promissora que permite acessar o genoma desses organismos incultiváveis, pois consiste na extração de DNA diretamente do ambiente e construção de uma biblioteca com este genoma misto. Neste trabalho descrevemos a construção de uma biblioteca feita com DNA de alto peso molecular isolado diretamente de solo coletado sob arboreto de eucaliptos no Estado de São Paulo, Brasil. A biblioteca possui 9.320 clones e foi construída em vetor cosmídeo, com insertos de tamanho variando entre 30 e 45kb... / Secondary metabolites are bioactive compounds with great importance in the pharmaceutical and agriculture industries, procuced by a few groups of microrganisms and plants. The polyketides that are synthetized by enzimatic complexes, denominated polyketides synthases, outstand among the secondary known metabolites, which are part of the main structure of many antibiotics. Ali genes involved in the biosynthesis of antibiotics are found as clusters in the chromossome. The traditional methods for the research of new drugs that are made from microrganisms cultures isolated from the soil are not so promissing, due to the high rate of rediscorevy of already known species, reaching 99.9%. The other small piece of microrganisms are culturable by standards culture methods, reaching 1 % maximum. Metagenomics is a promissing approach that allows the access to genom of these organisms that are not culturable, as it is carried out by DNA extraction directly from the environment and construction of a mixed genomic library. In this work, we describe the construction of a library made from high molecular weight DNA isolated directly form the soi! undemeath a pinus forest in the State of São Paulo, Brazil. The library shows 9.320 dones and it was constructed in a cosmideo vector, with insert size ranging from 30 to 45 kb. Digestion with difterent restriction enzymes of cosmidial DNA randomly chosen allowed to visualize evident difterences in the restriction fragments among the clones, as does the possibility to determine the average insert size. The initial evaluation of the presence of genes involved in the biosynthesis of antibiotics synthesized by the enzymatic system PKS of kind I, was accomplished by the PCR amplification of clones from the library using specific primers. We studied 4.320 clones and the results suggest a great variety of these genes. The PCR products obtained were sequenced for the determination of identity of the amplified gene.

Made available in DSpace on 2014-06-11T19:28:23Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-18Bitstream added on 2014-06-13T19:36:51Z : No. of bitstreams: 1 borges_ll_me_jabo.pdf: 1223418 bytes, checksum: 842f71b580aef2f2202cf5ed68a06a43 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Foi realizado um experimento com frangos de corte machos e fêmeas, com duração de 43 dias, no Aviário Experimental do Departamento de Zootecnia da Faculdade de Ciências Agrárias e Veterinárias, UNESP, Campus de Jaboticabal, SP. O objetivo foi o de avaliar a eficácia de três programas de alimentação contendo virginiamicina, sobre o desempenho, rendimento de carcaça e morfometria intestinal de frangos de corte machos e fêmeas, criados sobre cama reutilizada. Foram utilizados 2.160 pintos de um dia de idade da linhagem Cobb, distribuídos em 72 boxes com 30 aves cada. O delineamento experimental adotado foi em blocos com esquema fatorial 3x2 (três programas alimentares e dois sexos), com doze repetições de 30 aves cada. As dietas experimentais consistiram em: T1 (dieta inicial, crescimento e final, isentas de virginiamicina), T2 (adição de virginiamicina nas dietas inicial, crescimento e final) e T3 (adição de virginiamicina nas dietas inicial e crescimento). Aos 21, 35 e 42 dias de idade foram avaliados os dados de desempenho (peso médio, ganho de peso, consumo de ração, conversão alimentar e viabilidade criatória) e aos 42 dias de idade as características de carcaça (rendimento de carcaça, peito, peito desossado, coxa + sobrecoxa e asas). Aos 43 dias de idade, amostras de duodeno e jejuno foram retiradas para análise de morfometria de altura e largura das vilosidades, profundidade de cripta e relação a altura de vilosidade/profundidade de cripta, no Laboratório de Histologia e Embriologia do Departamento de Morfologia e Fisiologia Animal da FCAV, UNESP. Os tratamentos T2 e T3 proporcionaram melhores resultados para peso, ganho de peso e conversão alimentar, no entanto, não interferiram no consumo de ração, viabilidade criatória e no rendimento de carcaça e cortes quando comparados com o tratamento T1. A utilização de virginiamicina proporcionou melhor... / An experiment was conducted with broiler males and females, with duration of 43 days in the Experimental Aviary of the Animal Science Department at Agriculture and Veterinary Sciences Faculty, UNESP, Jaboticabal, SP. The objective was to evaluate the efficacy of three feed programs containing virginiamycin on performance, carcass and intestinal morphology of broiler males and females reared on reused litter. We used 2,160 chicks a day old, Cobb, distributed in 72 boxes with 30 birds each. The experiment was arranged in blocks with a 3x2 factorial arrangement (three food programs and two sexes), with twelve replicates of 30 birds each. The experimental diets consisted of T1 (starter, growth and final diet free virginiamycin), T2 (addition of virginiamycin in diets starting, growing and finishing) and T3 (addition of virginiamycin in diets and initial growth). At 21, 35 and 42 days of age were evaluated performance data (average weight, weight gain, feed intake, feed conversion and livability) and at 42 days old carcass characteristics (carcass yield, breast, boneless breast, thigh + drumstick and wings). At 43 days of age, samples of duodenum and jejunum were removed for morphometric analysis of villus height and width, crypt depth and compared villus height/crypt depth in the Histology and Embryology Laboratory, of Morphology and Physiology Departament, from FCAV, UNESP. The treatments T2 and T3 provided better results for weight, weight gain and feed conversion, however, did not affect feed intake, livability and carcass yield and cuts when compared with T1. The use of virginiamycin provided better development of the villi and crypts, when compared with treatment without antibiotic in the study