Submitted by Celso Magalhaes (celsomagalhaes@ufrrj.br) on 2018-08-29T13:47:37Z No. of bitstreams: 1 2009 - Cl?udia de Andrade Moura.pdf: 1773774 bytes, checksum: 32b0c84e574fc6692f8b7bcf05b86038 (MD5) / Made available in DSpace on 2018-08-29T13:47:37Z (GMT). No. of bitstreams: 1 2009 - Cl?udia de Andrade Moura.pdf: 1773774 bytes, checksum: 32b0c84e574fc6692f8b7bcf05b86038 (MD5) Previous issue date: 2009-12-16 / The educational work does not involve only the transmission of the knowledge, but the pedagogic and political competence, where learning ove rlaps teaching and the student is the learning agent. The aim of this study was to evaluate the reflexes of the application of different teaching methodologies and educational games in the appropriation of the knowledge in relation to the contents: Good Manufacturing Practices (GMP) and Sanitation Standard Operating Procedures (SSOP), worked in the Agricultural Technical Course through swine slaughtering practical classes, carried at the Federal Technological University of Paran? (UTFPR). The evaluation instrument was a multiple choice questionnaire, applied in classroom at four different moments: a) Diagnosis; b) intervention I (traditional theoretical and practical lessons); c) intervention II (?innovative? slaughtering, with GMP); d) intervention III (educational game application). Along the development of the intervention processes the pupils were getting conscious about cares within slaughtering. The results of the microbiological and physical-chemical analysis of meat, hands of the manipulators and instruments surfaces sampled during the traditional (T1) and the correct mode of slaughtering (T2) were compared. The first one (T1) proved to be inadequate and does not attend the minimum requirements for safety assurance. The significance of the differences in the numbers of correct answers between the different moments of application of the questionnaire were evaluated by t-Students test, as paired data, and presented as boxplot graphs, in lines and index numbers. A significant at 5% (p<0,05) increment was observed at all stages of the educative process. The percentage of correct answers at the third evaluation was more than 70% as compared to the diagnosis moment, confirming that all the stages had been important in the knowledge achievement ; especially through real data evidenced by the pupils. / O trabalho docente n?o s? envolve a transmiss?o de conhecimento, mas a compet?ncia pedag?gica e pol?tica, onde o aprender sobrep?e o ensinar, e o aluno ? o agente desse aprendizado. Assim, o objetivo deste estudo foi averiguar se diferentes metodologias de ensino, bem como a aplica??o de jogos educativos refletem na apropria??o do conhecimento em rela??o aos conte?dos: Boas Pr?ticas de Fabrica??o (BPF) e Procedimento Padr?o de Higiene Operacional (PPHO), trabalhados no curso t?cnico em agropecu?ria, dentro do processo tecnol?gico de abate su?no, realizados na Universidade Tecnol?gica Federal do Paran? (UTFPR). O instrumento de avalia??o utilizado foi um question?rio de m?ltipla escolha, aplicado em sala de aula em quatro momentos diferentes: a) Diagn?stico; b) interven??o I (aula te?rica e pr?tica tradicional); c) interven??o II (abate ?inovador?, BPF); d) interven??o III (aplica??o do jogo educacional). Com o desenrolar do processo de interven??es ouve uma percep??o dos alunos no que diz respeito ao processo de abate. Foram comparados os resultados das analises microbiol?gicas e f?sico-qu?micas da carne e de superf?cies de instrumentos e m?os de manipuladores durante o abate na forma tradicional (T1) com os obtidos ap?s o abate com interven??o (T2). O primeiro mostrou-se inapropriado por n?o atender os requisitos m?nimos exigidos para obten??o de produto seguro. Para verifica??o de diferen?as significativas no n?mero de acertos nas avalia??es em cada etapa, utilizou-se o teste de hip?tese t-Student, para dados pareados e apresentados em gr?ficos boxplot, em linhas e n?meros ?ndices. Observou-se um ganho crescente durante todas as etapas do processo educativo, significativo ao n?vel de 5% (p<0,05). O aumento das percentagens de acerto, ap?s a 3? avalia??o, em rela??o ? avalia??o diagn?stico, foi maior que 70%, confirmando que todas as etapas foram importantes na constru??o do conhecimento, principalmente atrav?s de dados reais vivenciados pelos alunos

製藥產業的屬性為民生必需工業，具有產品需求彈性低之特質，同時由於藥品影響人類健康，各國莫不希望引進具療效且安全性高之新藥，因此許多先進國家之藥廠，均以全球市場為腹地，積極推出新藥並申請各國專利，進行跨國行銷，所以全球製藥產業的發展，可視為一場國際競爭，而台灣的政府與製藥廠商也希望在全球製藥業之採購鍊上，取得適當卡位。近幾年，政府針對高科技產業，包括：製藥產業之發展，所必備的資本要素，提出各項重大改革，例如：民國86年主張「修訂第三類上市股票及上櫃股票科技事業審查要點」，民國89年實施「櫃檯買賣第二類股票制度」，對製藥產業資金的挹注很有助益。 資金的挹注雖是產業成長的契機之一，但由於製藥產業動態成長與不斷的演進，現有的法規、制度和產業環境，還需要政府適時修改不合時宜的法令，並規畫一個適合「製藥廠商」進行創新產業環境，同時廠商本身也應努力取得各項資源並有效使用，期使企業績效提昇能反映在製藥產業績效與國際競爭力上。換句話說，製藥產業內，希望取得大眾資金挹注之準上市（櫃）公司，如何藉由上市取得資本優勢，再轉換成企業競爭優勢以提昇經營績效應是燃眉之急。本研究認為準上市（櫃）藥廠，可借助已上市製藥廠，包括：中化、永信、生達與葡萄王之經驗，擷長補短以擬定適當的經營策略，相信對企業績效的提昇和獲得投資者的青睞應有正面助益。 所以本論文以已上市的製藥廠之經營策略發展趨勢和經營績效分析為研究主題，並同時比對政府政策、製藥廠商的策略作為趨勢及產業績效分析，試圖完整反應上市藥廠於後GMP時期（民國76年產業全面實施GMP認證制度後，至今）之經營動態，實證分析發現： 一、後GMP時期，中化、永信、生達之經營策略作為，具顯著的策略同形，且與製藥產業的發展趨勢相近。 二、後GMP時期之中化、永信、生達，三者之策略規畫，出現「同中有異」之現象，而葡萄王發展出截然不同的策略走向。 三、後GMP時期，政府根據製藥產業的需求，增修相關產業政策，中化、永信、生達也積極順應產業政策之變化，調整企業的各項經營策略。 四、永信、生達之營業成長率、投資報酬率、資本報酬率、固定資產周轉率、總資產周轉率佳，反應永信、生達的成長力、獲利力與經營效能良好；另一方面，中化則因過度使用財務槓桿、固定資產周轉率不佳及其轉投資大陸之獲利尚未能回補所投入的成本，形成其經營的困境，而葡萄王之營運體質雖良好，但因近幾年的獲利狀況不佳，稅後淨利在84、85年即低於產業水準，導致股價的表現疲軟。 五、根據第四項的財務指標綜合顯示，永信與生達是已上市藥廠間，其於成長力、獲利力與經營效能方面表現優異的廠商，印證其個別的經營策略發展趨勢，本研究推論下列策略目標有助於製藥廠商達到提高成長力、獲利力與經營效能之績效目標： 1. 開發自有品牌 2. 重視技術移轉，並積極與國內、外的學、研發機構或同業技術合作。 3. 不使用過高的財務槓桿，追求成長。 4. 企業應在製藥本業上追求專業化。 5. 謹慎評估大陸投資，定期評估公司的風險。 六、根據多元回歸分析，發現上市藥廠的毛利率、固定資產周轉率、總資產周轉率、研發費用佔銷售額的比率、應付帳款周轉率會影響上市藥廠之稅前淨利率、每股盈餘及總資產報酬率。亦即，上市藥廠雖具有規模（資本與營收）的優勢，但是企業是否能獲利，主要與其經營效能、研發投入、毛利率及是否取得供應商的信用融資相關。

Nous étudions comment faciliter la réutilisation des <br />preuves formelles en théorie des types. Nous traitons cette question <br />lors de l'étude <br />de la correction du programme de calcul de la racine carrée de GMP. <br />A partir d'une description formelle, nous construisons <br />un programme impératif avec l'outil Correctness. Cette description <br />prend en compte tous les détails de l'implantation, y compris <br />l'arithmétique de pointeurs utilisée et la gestion de la mémoire. <br />Nous étudions aussi comment réutiliser des preuves formelles lorsque<br />l'on change la représentation concrète des données. <br />Nous proposons un outil qui permet d'abstraire <br />les propriétés calculatoires associées à un type inductif dans<br />les termes de preuve.<br />Nous proposons également des outils pour simuler ces propriétés<br />dans un type isomorphe. Nous pouvons ainsi passer, systématiquement,<br />d'une représentation des données à une autre dans un développement<br />formel.

Les législations européennes imposent aux constructeurs automobiles que leurs véhicules ne dépassent pas un certain niveau de bruit. De plus, du point de vue économique, leurs clients ac- cordent de plus en plus d'importance aux aspects liés à la qualité acoustique lors de l'achat d'une voiture. Une des sources principales de bruit est le Groupe Moto Propulseur. Or, si des outils suc- cincts existent très en amont dans la conception, les concepteurs ne possèdent pas d'outils in- formatiques adaptés pour orienter la conception et ont donc peu de marge de manœuvre. Si le moteur s'avère bruyant en fin de projet, seules des solutions palliatives coûteuses et souvent mal adaptées peuvent alors être appliquées. L'objectif de cette thèse est par conséquent de permettre d'évaluer le bruit émis par le moteur plus tôt dans de cycle de conception grâce à des simulations numériques. Ces simulations doivent être effectuées sur des pièces de géométrie simplifiées conformément au niveau de connaissance à ce stade de la conception. De plus, des méthodes numériques rapides doivent permettre de com- parer plusieurs architectures moteur afin de privilégier la plus favorable par rapport à l'acoustique et aux vibrations. Pour y parvenir, nous avons déterminé les paramètres géométriques correspondant à un stade précoce de la conception qui doivent apparaître sur les pièces simplifiées afin d'obtenir des simu- lations représentatives vis-à-vis de la vibroacoustique. Dans ce but, nous avons mis en place une méthodologie de représentation simplifiée juste nécessaire correspondant à ces prestations. Elle a été évaluée en dynamique d'une part et en acoustique d'autre part. De plus, il est indispensable de disposer de nouvelles méthodologies de calcul du rayonne- ment acoustique, adaptées à la comparaison d'architectures. Pour cela, nous avons développé la Sous Structuration Soustractive (SDM) qui permet de décomposer un problème complexe de rayonnement acoustique en deux sous problèmes, l'un fini et l'autre infini. En choisissant de fa- çon adéquate cette décomposition lorsqu'on souhaite comparer plusieurs architectures moteur, cette méthode permet de conserver les résultats du calcul infini souvent traité avec les éléments de frontières. Les résolutions des sous problèmes finis résultants sont alors effectuées par une mé- thode éléments finis. Le calcul infini utilisant les éléments de frontière étant plus long à réaliser, nous avons également proposé une méthode appelée Sous Structuration Infinie ou Algorithme de clonage qui permet de traiter ce type de problème en utilisant les éléments finis. Enfin la SDM a été appliquée à un cas industriel. Les résultats numériques ont été comparés avec succés à ceux obtenus avec une méthode de référence.

El cisplatí, PtCl2(NH3)2, ha estat una de les drogues més utilitzades en la quimioteràpia del càncer des del descobriment de la seva activitat. Però degut a la seva alta toxicitat i greus efectes secundaris, s'han sintetitzat nous compostos amb la finalitat de reduir aquests inconvenients.En aquest sentit, el treball desenvolupat en aquesta tesi doctoral ha estat la síntesi i caracterització de tretze complexos de Pt(II) amb la finalitat d'estudiar llur activitat antitumoral. Aquests complexos presenten unes característiques estructurals comunes: geometria cis, dos lligands làbils de tipus clorur i un lligand diaminoquelatant derivat dels àcids d,l-2,3-diaminopropiònic (Hdap) i d,l-2,4-diaminobutíric (Hdab). S'han dissenyat unes estratègies sintètiques a partir de les quals els lligands han estat funcionalitzats amb diferents grups de tipus éster, aminoàcid i peptídic: Etdap·2HCl, Etdab·2HCl, [(dap-Metala)·2CF3COOH], [(dab-Metala)·2CF3COOH], [(dap-phe)·2CF3COOH], [(dab-phe)·2CF3COOH], [(dap-Mettrp)·2CF3COOH], [(dab-Mettrp)·2CF3COOH], [(dap-ASTTTNYT-NH2)·2CF3COOH], essent Metala= éster metílic de L-alanina, phe= L-fenilalanina, Mettrp= éster metílic del L-triptofà. Aquests lligands diaminoquelatants s'han utilitzat per sintetitzar els corresponents complexos de Pt(II): PtCl2(Hdap), PtCl2(Hdab), PtCl2(Etdap), PtCl2(Etdab), PtCl2(dap-Metala), PtCl2(dab-Metala), PtCl2(dap-ala), PtCl2(dab-ala), PtCl2(dap-phe), PtCl2(dab-phe), PtCl2(dap-Mettrp), PtCl2(dab-Mettrp), PtCl2(dap-ASTTTNYT-NH2).A través de diferents tècniques i assaigs biològics (dicroisme circular, electroforesi en gel d'agarosa, microscopia de forces atòmiques, citometria de flux, assaigs de proliferació cel·lular) s'ha pogut demostrar l'activitat antitumoral d'aquests compostos. A través de la tècnica de dicroisme circular (DC) s'ha pogut demostrar que els lligands lliures no interaccionen covalentment amb el DNA de Calf Thymus i no modifiquen l'estructura secundària de la doble hèlix. En canvi, els respectius complexos han demostrat tenir capacitat per interaccionar amb el DNA i modificar la seva estructura secundària. Els complexos PtCl2(Hdap), PtCl2(Hdab) i PtCl2(dab-phe) mostren un comportament similar al cisplatí, generant adductes cis-bifuncionals que distorcionen la doble hèlix de forma no desnaturalitzant amb obertura de la doble cadena. Els complexos PtCl2(Etdap), PtCl2(Etdab), PtCl2(dap-ala), PtCl2(dab-ala), PtCl2(dap-Metala), PtCl2(dab-Metala), PtCl2(dap-phe), PtCl2(dap-ASTTTNYT-NH2) quan interaccionen amb el DNA generen un canvi en la conformació del DNA de la forma B a la forma C, produint-se un augment de la curvatura de l'hèlix per rotació de les bases nitrogenades. En aquests estudis s'ha comprovat que l'estructura del complex influeix en l'efecte generat sobre l'estructura secundària de l'àcid nucleic. En primer lloc, existeix una diferència en el comportament en funció del tamany del lligand diaminoquelatant, de manera que els complexos amb el lligand (dab) provoquen un efecte més remarcable. També s'observa aquest canvi de comportament al passar dels complexos que tenen el grup funcional esterificat als que el tenen protonat. D'aquesta manera, s'observa un major efecte sobre l'estructura secundària del DNA en aquells complexos que tenen el lligand diaminoquelatant de tres metilens (dab) i amb el grup carboxilat terminal protonat.Per tal de modelitzar la interacció d'aquests complexos amb el DNA, s'ha estudiat la interacció d'aquests compostos de Pt(II) amb 5'-GMP a través de RMN-1H, observant la variació dels senyals corresponents al H8 de 5'-GMP. Així s'ha pogut demostrar que aquests compostos interaccionen amb la 5'-GMP a través d'un enllaç covalent Pt-N7, de la mateixa manera a com interacciona el cisplatí.A través d'electroforesi en gel d'agarosa i microscopia de forces atòmiques (AFM) s'ha pogut determinar l'efecte que generen els lligands lliures i els respectius complexos de Pt(II) sobre l'estructura terciària del plasmidi pBR322. Els lligands provoquen un augment de l'agregació de les molècules de DNA i un lleuger augment de la compactació de l'estructura terciària. Aquests resultats s'atribueixen a la capacitat d'aquests compostos a interaccionar per pont d'hidrogen amb el DNA. Els corresponents complexos de Pt(II) provoquen un augment de l'agregació i una important compactació, degut per una banda a la capacitat de l'àtom de Pt a interaccionar covalentment amb el DNA, i per altra banda, a la capacitat del lligand a interaccionar per pont d'hidrogen amb l'àcid nucleic.Finalment s'ha estudiat l'activitat citotòxica d'aquests complexos de Pt(II) en diferents línies cel·lulars: A431 (línia de carcinoma epidermoide), HeLa (línia de carcinoma de coll d'úter) i HL-60 (línia promielocítica de leucèmia). Els complexos moderadament solubles en aigua, PtCl2(Hdap), PtCl2(Hdab), PtCl2(dap-ala), PtCl2(dab-ala), PtCl2(dap-phe) i PtCl2(dab-phe), han demostrat ser actius. L'activitat depèn de la concentració de complex, del temps d'incubació i de la línia cel·lular. Per temps d'incubació alts i concentracions de complex elevades s'observa la màxima activitat. Els complexos de l'alanina, PtCl2(dap-ala) i PtCl2(dab-ala), són els que mostren més activitat, mentre que els compostos de la fenilalanina són els menys actius, degut probablement a la voluminositat del lligand, la qual pot impedir o dificultar el transport del compost a través de la membrana cel·lular.L'activitat citotòxica dels complexos insolubles en aigua, PtCl2(Etdap) i PtCl2(Etdab), queda bloquejada per l'elevada concentració de DMSO (12%) necessària per solubilitzar els compostos. Aquests resultats permeten deduir que la presència d'un 12% de DMSO anul·la l'activitat d'aquests complexos, ja que el DMSO pot coordinar-se amb el Pt ocupant les posicions làbils del complex i evitant que es pugui coordinar amb el DNA.Els assaigs de proliferació cel·lular del complex PtCl2(dap-ASTTTNYT-NH2) i del pèptid lliure ASTTTNYT-NH2 han demostrat que ambdós compostos són actius. Tot i això, l'activitat del complex és superior a la del pèptid lliure, ja que el Pt pot interaccionar covalentment amb el DNA i augmentar l'efecte citotòxic. Per tant, el complex presenta un lligand portador biològicament actiu que pot transportar el metall a través de la membrana cel·lular i facilitar així la seva interacció amb el DNA.A través de la tècnica de citometria de flux s'ha comprovat que en tots els casos la mort cel·lular produïda pels complexos ha estat per apoptosi.Per últim, s'ha sintetitzat i caracteritzat un complex trinuclear de Pt(II), {[Pt(Me2Bpy)2][PtCl2(Me2Bpy)]2}, essent Me2Bpy= 4,4'-dimetil-2,2'-dipiridil. La resolució de la seva estructura per difracció de Raig-X ha permès determinar l'existència d'una interacció intramolecular Pt-Pt de 3.474 Å. / Cisplatin, PtCl2(NH3)2, has been one of the most used drugs for the cancer chemotherapy. However, due to its high toxicity and important side effects, new complexes have been synthesized in order to reduce these disadvantages.In that way, the work presented in this thesis is the synthesis and characterization of 13 Pt(II) complexes in order to study their antitumor activity. These complexes present some common traits: cis geometry, two labile chloride ligands, and one diamine chelating ligand derived from d,l-2,3-diaminopropionic acid (Hdap) and d,l-2,4-diaminobutyric acid (Hdab). These ligands have been derived using new synthetic strategies with different functional groups as ester, aminoacid and peptide type: Etdap·2HCl, Etdab·2HCl, [(dap-Metala)·2CF3COOH], [(dab-Metala)·2CF3COOH], [(dap-phe)·2CF3COOH], [(dab-phe)·2CF3COOH], [(dap-Mettrp)·2CF3COOH], [(dab-Mettrp)·2CF3COOH], [(dap-ASTTTNYT-NH2)·2CF3COOH], being Metala= methyl ester of L-alanine, phe= L-phenylalanine and Mettrp= methyl ester of L-triptophan. These ligands have been used for preparing the following Pt(II) complexes: PtCl2(Hdap), PtCl2(Hdab), PtCl2(Etdap), PtCl2(Etdab), PtCl2(dap-Metala), PtCl2(dab-Metala), PtCl2(dap-ala), PtCl2(dab-ala), PtCl2(dap-phe), PtCl2(dab-phe), PtCl2(dap-Mettrp), PtCl2(dab-Mettrp), PtCl2(dap-ASTTTNYT-NH2).Using different techniques and biologic assays (circular dichroism, agarose gel electrophoresis, atomic force microscopy, flow cytometry, cell proliferation assays) the antitumor activity of these complexes has been demonstrated.With circular dichroism it has been demonstrated that free ligands do not covalently bind Calf Thymus-DNA and do not modify its secondary structure. In constrast, the corresponding Pt(II) complexes are able to interact with DNA and modify its secondary structure. Complexes PtCl2(Hdap), PtCl2(Hdab) and PtCl2(dab-phe) behave in the same direction as cisplatin, forming cis-bifunctional intrastrand adducts that probably open the double helix. The other complexes, PtCl2(Etdap), PtCl2(Etdab), PtCl2(dap-ala), PtCl2(dab-ala), PtCl2(dap-Metala), PtCl2(dab-Metala), PtCl2(dap-phe), PtCl2(dap-ASTTTNYT-NH2), interact with DNA generating a B&#61614;C transformation with increasing winding of the double helix by rotation of the bases. The influence of the complex structure in the effect on the secondary structure of DNA has been proved. Firstly, a difference in the behaviour exists depending on the ring size, so complexes with (dab) ligand cause bigger modifications. This kind of behaviour is also seen between complexes with ester groups at the C-terminus and complexes with carboxylic group. Thus, there is a greater effect in the secondary structure of the DNA in those complexes that have the (dab) ligand and a protonated carboxylate group.The interaction between the platinum complexes and 5'-GMP has been studied by 1H-NMR, following the variation of H8 signals from 5'-GMP. It has been demonstrated that all complexes covalently interact with 5'-GMP through the N7 of the base, as cisplatin do.The effect generated by free ligands and their Pt(II) complexes in the tertiary structure of pBR322 DNA has been determined by agarose gel electrophoresis and atomic force microscopy (AFM). Generally, the free ligands studied generate an increase of DNA aggregation and a slight coiling of the double helix, due to their possibility of forming hydrogen bonds with the DNA bases through different functional groups of their structure. The Pt(II) complexes generate an important coiling and aggregation of DNA, probably due to the covalent binding Pt-DNA and hydrogen bond interactions through their ligands.The cytotoxic activity of the Pt(II) complexes has been evaluated through cell proliferation assays in A431, HeLa and HL-60 cell lines using different incubation times (24h, 48h, 72h). The water-soluble complexes, PtCl2(Hdap), PtCl2(Hdab), PtCl2(dap-ala), PtCl2(dab-ala), PtCl2(dap-phe) i PtCl2(dab-phe), show antitumor activity in a dose- and time- dependent manner, gradually decreasing the cell survival percentage when the complex concentration increase, in the same direction as carboplatin. All these complexes present the highest activity at long incubation times and high complex concentration. The alanine complexes, PtCl2(dap-ala) and PtCl2(dab-ala) are the most active and those of phenylalanine are the less active, probably due to the ligand size, which can make its transport through cell membrane more difficult.The water-insoluble complexes, PtCl2(Etdap) and PtCl2(Etdab), apparently do not show antitumor activity due to the presence of 12% DMSO necessary por solving these complexes. In these conditions they do not cause a decrease in cell survival. The DMSO could interact with platinum, displacing the labile ligands of the complex and not allowing it to interact with DNA. Complex PtCl2(dap-ASTTTNYT-NH2) is also insoluble in water, but can be dissolved in 2% DMSO solution. Cell proliferation assays show that the free peptide, ASTTTNYT-NH2, and the platinum complex are both active. The activity of the complex is higher than the free peptide, probably because platinum can covalently interact with DNA and increase the cytotoxic effect. This result suggest that the ligand can act as a carrier ligand and make the interaction metal-DNA be easier.Flow cytometry assays have demonstrated that cell death generated by the complexes synthesized is apoptosis. Agarose gel electrophoresis of extracted DNA show a classical 200-base pair integer oligonucleosome ladder.A new trinuclear Pt(II) complex, {[Pt(Me2Bpy)2][PtCl2(Me2Bpy)]2} being Me2Bpy= 4,4'-dimethyl-2,2'-dipyridyl, has been synthesized and characterized. The X-ray diffraction of its structure shows the existence of an intramolecular Pt-Pt interaction of 3.474 Å.

Les Escherichia coli entérohémorragiques (EHEC) sont responsables de toxi-infections alimentaires conduisant à des colites hémorragiques pouvant se compliquer d'un syndrome hémolytique et urémique. Une fois arrivés dans l'intestin, les EHEC adhèrent aux cellules épithéliales en causant des lésions d'attachement-effacement. Le système de sécrétion de type III et les protéines effectrices requis pour ce phénotype sont codés majoritairement par le locus d'effacement des entérocytes (LEE), constitué de plusieurs opérons (LEE1-5). Notre étude a permis de clarifier une des cascades de régulation contrôlant l'expression du LEE. Par des analyses en qRT-PCR et des immuno précipitations de la chromatine, nous avons déterminé que les régulateurs GadE et GadX sont des répresseurs indirects de l'expression du LEE. GadE active l'expression de gadX, et GadX réprime l'expression de ler, codant pour le principal activateur des opérons LEE2-5. De plus, GadE réprime aussi l'expression des opérons LEE4 et LEE5 indépendamment de Ler. En retour, Ler réprime l'expression de gadE et de gadX. Le monoxyde d'azote (NO) est un effecteur majeur de la réponse immune innée, produit en particulier par les cellules épithéliales intestinales. Il avait été montré que le NO réprime l'expression du LEE et active celle de gadE et de gadX. Notre étude a permis d'identifier le régulateur clé responsable de ces régulations, NsrR. NsrR réprime indirectement l'expression de gadE et gadX et active l'expression des opérons LEE1, LEE4 et LEE5 en se fixant sur leurs promoteurs respectifs. En présence de NO, NsrR devient inactif. Ainsi, le NO réprime directement l'expression du LEE en supprimant la fixation de NsrR aux promoteurs du LEE1, LEE4 et LEE5, et indirectement en activant l'expression de gadE et donc de gadX. Un modèle de régulation intégrant l'ensemble de ces résultats est proposé. D'autre part, nous avons identifié et caractérisé une nouvelle phosphodiestérase spécifique des EHEC les plus pathogènes, VmpA. Par son activité d'hydrolyse du di-GMPc, VmpA contrôle la mobilité bactérienne, la formation de biofilm, et probablement l'expression du LEE, mais aurait aussi un rôle plus général dans la physiologie des EHEC.

Bacterial stationary phase is an interesting biological system to study, as the organism undergoes several metabolic changes during this period and new molecules are generated to support its survival. The stationary phase of mycobacteria has been extensively studied since the discovery of Mycobacterium tuberculosis, the causative agent of tuberculosis. The stationary phase of mycobacteria adds further complication as many antibacterial drugs become less effective. The M. tuberculosis infects the alveolar macrophages and dendritic cells or monocytes recruited from peripheral blood. Macrophages are supposed to provide an initial barrier against the bacterial infection, but fails. Mycobacteria have evolved several strategies to survive and set up an initial residence within these cells and grow actively inside the host. The host immune system tries to limit the bacterial growth and confines the organism to a latent state in which the organism can persist indefinitely, known as granuloma stage. During latency or granuloma stage mycobacteria can retain the ability to resume the growth in the future. Mycobacteria must adapt to a highly dynamic and challenging environment because the interior environment of granuloma is devoid of or in low level of oxygen, depleted nutrient, high carbon dioxide, and possess increased levels of aliphatic organic acids and hydrolytic enzymes. The survival of a bacterium in less nutrient supply or in depleted oxygen is important for its long-¬term persistence inside the host under harsh environmental conditions. Mycobacterium smegmatis is the closest non-¬pathogenic homologue of M. tuberculosis, and has been used widely as a model system to study gene regulation under such conditions. In these harsh environmental conditions bacteria need to sense the external environment to modulate their gene expression. More importantly, each individual cell should communicate with its neighbours, and the response takes place in a concerted manner, which is termed as quorum sensing. Thus, the quorum sensing is a cell-¬cell signaling process that allow the bacteria to monitor the presence of other bacteria in their surroundings by producing and responding to small signaling molecules, which are known as autoinducers. It is a density dependent phenomenon and regulates the expression of the genes in response to fluctuation in cell¬-population density. A minimum threshold level of autoinducers is necessary to detect the signal and respond to it. Quorum sensing enables bacteria to behave like multicellular organisms and controls group activities like biofilm formation, sporulation, bioluminescence, virulence, and pigment production, etc (Bassler, 1999; Camilli & Bassler, 2006; Fuqua et al., 1996; Miller & Bassler, 2001). In Gram-¬negative bacteria, small-¬molecules, which are known as autoinducers are produced. They are acyl homoserine lactones (AHLs), which are derived from S¬adenosyl methionine (SAM) and particular fatty acyl carrier protein by LuxI¬type AHL synthases (Fuqua et al., 1996). In Gram-¬positive bacteria small peptides autoinducers, 5¬12 amino acids long, play an active role in communication. These oligopeptides are post--translationally modified by the incorporation of lactone and thiolactone rings, lanthionines and isoprenyl groups. These oligopeptide autoinducers are detected by membrane-¬bound two-¬component signaling proteins, and signal transduction occurs by a phosphorylation cascade (Camilli & Bassler, 2006; More et al., 1996; Novick, 2003; Zhang et al., 2002). In bacteria, the cyclic adenosine monophosphate (cAMP), and guanosine pentaphosphate and/or tetraphosphate ((p)ppGpp) are well known second messengers, which play important role in relaying extracellular information, but recently cyclic diguanosine monophosphate (c-¬di¬-GMP) is being studied most comprehensively as a nucleotide-¬based second messenger. C-¬di¬-GMP was first discovered in Gluconacetobacter xylinus as a positive allosteric regulator of cellulose synthase (Ross et al., 1987; Tal et al., 1998; Weinhouse et al., 1997). The in vivo level of c-¬di-¬GMP in bacterial cell is maintained by the balance between diguanylate cyclase and phosphodiesterase activities. The GGDEF and EAL amino acids sequence are the signature motif for GGDEF and EAL domain protein within its active site, respectively. The GGDEF domain protein is involved in synthesis of c-¬di-¬GMP and the EAL domain protein is involved in the hydrolysis of c-¬di-¬GMP, and the majority of these proteins contain additional signal input domains (Paul et al., 2004; Ross et al., 1987; Ryjenkov et al., 2005; Tal et al., 1998). M. smegmatis has a single bi-¬functional protein having both the domains, GGDEF and EAL, for the diguanylate cyclase (DGC) and phosphodiesterase (PDE¬A) activities. In addition to GGDEF and EAL domain, one sensory domain, GAF, is also there at the N-terminal of MSMEG_2196 in M. smegmatis. In the present investigation, studies have been carried out to understand the regulation of c-¬di-¬GMP in M. smegmatis at protein and gene level. The entire study on mycobacterial MSMEG_2196 (msdgc¬1) can be broadly divided into five parts; the first part will cover the identification and biochemical characterization of MSDGC¬1 protein, responsible for the regulation of in vivo c-¬di-¬GMP concentration in M. smegmatis, and the presence of GGDEF¬EAL domain containing proteins in various mycobacterial species. The second part will cover the structure function relationship as a function of substrate, GTP and product, c-¬di-GMP, molecule using fluorescence spectroscopy as a tool, and the mutational and structural studies, which leads to the identification of a novel structural motif. The third part will cover the characterization of msdgc¬1 gene knockout and complementation studies in great detail. The fourth part will comprise in vivo and in vitro promoter characterization and regulation of the msdgc¬1 gene under nutritional starvation. The last chapter will cover the characterization of novel synthetic glycolipids, which are working as a growth and biofilm inhibitors in mycobacteria, and can be used as a new drug candidates. Chapter 1 outlines the signal transduction and quorum sensing mechanism, and small molecule signaling modules in brief. The importance of the study started with a brief introduction about the historical aspect of tuberculosis, the current scenario of the treatment of tuberculosis. The urgent need for new drug targets and drugs will be discussed. The important role of the novel second messenger, c-¬di¬-GMP has been explained in greater details in both Gram-¬positive and Gram-¬negative bacteria, and the information available on the different cellular targets has been documented. Chapter 2 describes the identification and biochemical characterization of M. smegmatis MSMEG_2196 protein. The domain architecture and individual domain role have been studied. The MSMEG_2196 proteins consist of three domains, GAF, GGDEF and EAL in tandem, and individual role of each domain has been studied. The diguanylate cyclases containing GGDEF and phosphodiesterases containing EAL domains have been identified as the enzymes involved in the regulation of in vivo cellular concentration of c-¬di-¬GMP. GAF domain has been identified as a metal binding domain in other bacteria and may be playing a role in the regulation of synthesis and hydrolysis activities of c-¬di¬-GMP. The identification, cloning expression and purification of MSMEG_2196 and MSMEG_2774 have been discussed. We have reported that mycobacterial MSDGC¬1 protein has dual activity, which means that it can synthesize and hydrolyse c¬-di-¬GMP; and also full-¬length protein is necessary for its either of the activities. The synthesis and hydrolysis products, c-¬di-¬GMP and pGpG, of MSDGC¬1 protein have been identified and characterized using radiolabelled alpha [α¬32P]GTP and Matrix Assisted Laser Desorption/Ionization mass spectrometry (MALDI). The effects of temperature and pH on the activities of MSDGC¬1 have been studied. The circular dichroism studies show that the MSDGC¬1 protein is predominantly α¬helical in nature, and secondary structure does not alter upon GTP binding. The kinetic parameters for MSDGC¬1 protein have been calculated as a function of substrate, GTP. The protein, MSDGC¬1, exist as a monomer and a dimer in solution. The MSDGC¬1 protein has four cysteines, and we have shown here using mass spectrometric analysis that none of the cysteines is involved in the disulphide linkage. Chapter 3 deals with the structure-¬function relationship as a function of GTP and c¬-di-GMP molecules using fluorescence spectroscopy as a tool. In order to do so we have generated several cysteine mutants using site directed mutagenesis, and protein was labelled with thiol-¬specific fluorophores. The labelled protein was checked for its DGC and PDE¬A activities and specificity of labelling was confirmed using MALDI and radiometric analysis. The Fluorescence Resonance Energy Transfer (FRET) has been carried out to observe domain-¬domain interaction as a function of GTP and c¬-di-¬GMP. The bioinformatics, structural, and mutational analysis suggest that cysteine at 579 position is important for DGC and PDE¬A activities, and may be involved in the formation of a novel structural motif, GCXXXQGF, which is necessary for synthesis and degradation of c-¬di-¬GMP. Chapter 4 describes the construction of a deletion mutation of MSMEG_2196 gene in M. smegmatis. The strategy for the construction of the knockout strain has been shown and confirmation of the knockout event has been carried out using PCR and Southern hybridization. The effect of deletion of msdgc¬1 has been studied in great detail, and it was noticed that biofilm formation is not affected, but long-¬term survival is significantly compromised. It is hypothesized here that c-¬di¬-GMP is involved in the regulation of cell population density in mycobacteria. We have successfully detected the c-¬di¬-GMP in the total nucleotide extract using HLPC coupled with MALDI, and we have shown here that level of c-¬di-¬GMP increases many fold in the stationary phase of growth under nutritional starvation. Chapter 5 deals with the identification and characterization of the promoter element of msdgc¬1 in M. smegmatis. The study was undertaken to understand the mechanism of regulation at promoter level. We have observed here that msdgc¬1 promoter is starvation induced, and expression of msdgc¬1 increases many fold in the stationary phase under nutritional starvation. We have also tried to establish the link between the ppGpp and c-di¬-GMP signalling, and possible role of c-¬di-¬GMP in the regulation of cell population density have been discussed. Further, the +1 transcription start site has been identified using primer extension method. The putative ¬10 hexamer region for the RNA polymerase binding has been identified and confirmed using site-¬directed mutagenesis. It was found to be TCGATA, which is 14 bp upstream from the +1 transcription start site. The msdgc-1 promoter is specific for mycobacteria and does not function in E. coli. Moreover, we have identified the sigma factors, which regulate the msdgc¬1 promoter in growth phase dependent manner. Chapter 6 begins with the screening of synthetic glycolipids as a novel drug candidate. The different glycolipids have been tested for their effect on growth, biofilm formation, and sliding motility of M. smegmatis, and we have screened few of them, which were found to be effective in inhibiting the microbial growth, biofilm formation, and sliding motility. Chapter 7 summarizes the work presented in this thesis. Appendix: The protein sequences of MSDGC¬1 and MSDGC¬2, and the multiple sequence alignments of MSDGC¬1 protein have been documented. The FORTRAN program, which was used to calculate spectral overlap integral J, and the diagrams of the plasmids used in this study have been provided.

Este projeto tem por objetivo desenvolver um checklist de boas práticas de fabricação (BPF) adaptado ao público das agroindústrias familiares de pequeno porte (AFPP) produtoras de embutidos, de modo que sirva como ferramenta de auxílio na recomendação das AFPP ao SUSAF-RS (Sistema Estadual Unificado de Sanidade Agroindustrial Familiar e de Pequeno Porte do RS). Foram visitadas 20 agroindústrias situadas nas regionais da Emater de Lajeado e Soledade, onde foi aplicado o checklist de BPF original (BRASIL, 2002) bem como o checklist adaptado desenvolvido. As agroindústrias foram classificadas em G1, G2 e G3 conforme o percentual de conformidade (C). A proposta do checklist adaptado se justificou uma vez que o instrumento proposto apresentou resultado de agroindústrias classificadas com G1 (>75% C) significativamente superior em relação à classificação obtida das mesmas agroindústrias submetidas ao checklist original. O checklist adaptado mostrou que a maioria das agroindústrias foi classificada como pertencente ao G2 com média de conformidade de 59,2%. Somente 4 agroindústrias, além da unidade onde foi aplicado o piloto, obtiveram mais de 75% de C sendo classificadas como G1. Todas estas estão registradas em Serviços de Inspeção Municipais já aderidos ao SUSAF, e uma apresenta registro no Serviço de Inspeção Estadual. Além destas, 8 agroindústrias foram classificadas no G2 e 7 foram classificadas no G3. As principais não conformidades (NC) identificadas nas classificações G2 e G3 foram relacionadas à gestão documental (SG5) e falhas no quesito higiênico-sanitário (SG2), sendo que 5 das 8 agroindústrias classificadas como G2 apresentaram C superior a 65%. Nestes casos, a gestão documental foi o principal gargalo para o não atingimento dos 75% de C preconizado. Ressalta-se que das 7 agroindústrias classificadas como G3, 4 apresentaram índices preocupantes no quesito sanitário (SG2) com C inferior a 25%. O trabalho evidenciou que as AFPPs localizadas em municípios cujos SIMs não estão estruturados, atrelado à presença de RTs pouco atuantes, ocasionam baixo índice de BPF, pois carecem de informações técnicas e capacitação para implantação das mesmas. Percebeu-se que a realidade da AFPP é distinta à da indústria de larga escala. Por isso, parece justo que os dois segmentos sejam avaliados de forma diferenciada, justificando o propósito do projeto. / This project deals with the adaptation of a Good Manufacturing Practices (GMP) checklist for application in small farm agroindustry producing sausages. The objective will be to enable the proposed checklist to serve as an aid tool in the recommendation of this profile of agroindustries to SUSAF-RS (Unified State System of Agroindustrial Health and Small Porte of RS). Twenty agroindustries distributed in the regional municipalities of Emater de Lajeado and Soledade were visited, where the original BPF checklist (BRASIL, 2002) was applied as well as the adapted checklist. The agroindustries were classified in G1, G2 and G3 according to the percentage of compliance (C). The proposal of the checklist adapted to measure BPF in the scope of the small farm agroindustry was justified, since the proposed instrument presented a result of agroindustries classified with G1 (> 75% C) significantly higher than the classification obtained from the same agroindustries submitted to the original checklist. The adapted checklist showed that most agro-industries were classified as belonging to G2 with a mean of 59.2% compliance. Only 4 agroindustries, besides the unit where the pilot was applied, obtained more than 75% of C being classified as G1, therefore, with GMP implanted. All of these are registered in Municipal Inspection Services that have already joined the SUSAF. In addition, 8 agro-industries were classified in G2 and 7 were classified in G3. The main non-conformities (NC) identified in the G2 and G3 classifications were related to document management (SG5) and sanitary-sanitary (SG2) failures, with 5 of the 8 agro-industries classified in G2 presenting C above 65%. cases, document management was the main bottleneck for not achieving the 75% of C recommended. It is noteworthy that of the 7 agroindustries classified as G3, 4 presented worrying rates in the sanitary (SG2) with C less than 25%. The work can show that the small farm agroindustry s located in municipalities whose Inspcetion Local Service are not fully structured, coupled with the presence of weakly active professional suporting, present a low GMP index, since they lack technical information and capacity to implement the Manual of GMPs and SSOPs. It was perceived that the reality of small farm agroindustry is distinct from that of large-scale industry. Therefore, it seems fair that these two segments are evaluated in a differentiated way, justifying the purpose of the project.

Made available in DSpace on 2014-06-11T19:23:27Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-02-18Bitstream added on 2014-06-13T20:10:50Z : No. of bitstreams: 1 seixas_frf_me_sjrp.pdf: 308372 bytes, checksum: 79dc807bb402317ba0cb325f7c61e4fa (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / No Brasil, estima-se que, de cada cinco refeições, uma não é efetuada em casa, sendo a salada de maionese um dos alimentos comumente consumidos. Este produto alimentício é basicamente constituído por vegetais cozidos posteriormente adicionados de maionese. Portanto, além da microbiota presente nos vegetais, a falta de higiene dos manipuladores durante o seu preparo, bem como as condições de armazenamento do produto, faz com que os microrganismos possam vir a ocasionar deteriorações no alimento ou intoxicações alimentares nos consumidores. Neste trabalho as Boas Práticas de Fabricação (BPF) foram verificadas em 10 estabelecimentos produtores de saladas de maionese aplicando um check - list que se constituiu de 123 itens de verificação baseado na legislação vigente no país (Resolução da Diretoria Colegiada - RDC nº. 216 de 2004) e análise da qualidade microbiológica perante 57 amostras destas saladas comercializadas na cidade de São José do Rio Preto - SP. As análises microbiológicas foram realizadas em 2 etapas: a primeira em 30 amostras no período de junho, agosto e outubro de 2006, e a segunda etapa no período de fevereiro, abril e junho de 2007, após a aplicação do check - list e orientações para o correto manuseio desse alimento. Foram realizadas as seguintes análises: contagem de bolores e leveduras, Staphylococcus aureus, Número Mais Provável (NMP) de coliformes totais, termotolerantes, pesquisa de Escherichia coli e de Salmonella spp., bem como procedeu-se a identificação das leveduras isoladas e o teste de resistência frente a diferentes concentrações do conservante sorbato de potássio. De acordo com o check - list aplicado para verificação das BPF, 70,0% dos estabelecimentos apresentaram não conformidades superiores a 25,0%, sendo classificados como insatisfatórios para produção de alimentos... / The aim of this work was to verify the Good Manufacturing Practices (GMP) and the microbiologic quality of the salads added of mayonnaise marketed in São José do Rio Preto - SP, through the following analyses: check - list application, based on the legislation in force in the country, which classified the levels of suitability presented by the producers, counting of mould and yeast, Staphylococcus aureus, determining the Most Probable Number of total and heat resistant coliforms, Escherichia coli and Salmonella spp research, and proceeded to identification isolated yeast and resistance test to different concentrations of potassium sorbate conserver. According to the check - list applied to GMP verification, 70,0% of the establishments showed no conformities above 25,0%, being classified as unsatisfactory for food production. The obtained result for mould and yeast counting varied from 1,9 x 103 to 6,0 x 106.. The biggest number of mayonnaise salad contamination was caused by Staphylococcus aureus with variations of < 100 a 1,8 x 107 UFC/g. For The Most Probable Number determination of total and heat resistant coliforms a variation of < 3 a > 1100 NMP/g was verified. Escherichia coli was found in all samples positive for heat resistantt coliforms. Salmonella spp. was not found. From the ten establishments 4 (40%) improved the salads quality with mayonnaise added and 1 (10%) stopped this food production after inspection and the technicians of Sanitary Survellance guidance. In relation to the isolated yeasts, Debaryomyces hansenii var. fabryii species was the most frequent, in 19 cultures (76,0%) followed by Cryptococcus laurentii with 4 (16%), Arxula adeninovorans with 1 (4,0%). The species Arxula adeninovorans and Candida edax were not sensitive to potassium sorbate conserver. In relation to...(Complete abstract click electronic access below)

The World-wide Organization of health (OMS) indicates that amongst the disease of food origin, more than 60% of the cases elapse of inadequate techniques of processing, involving the pathogenic microorganisms and parasites, beyond its toxic products. When if it says in management of quality in the industrialization and food manipulation, is obligatory the program of Good Manufacture Practices (GMP), and Hazard Analysis and Critical Control Point (HACCP). In foods unit the quality and safety is related to the productivity. For the aspect relative security to the consumer, these instruments provide to influence concepts of quality and safety that are fiscalized and implemented in accordance with current law. For food unit is important that these also been engaged and sensitized of the importance to establish standards procedures hygienc, with sights to the sanitary hygienical conditions of the installations, equipment, utensils and manipulations hands. Provide to responsible and manipulations base technique to on the safe food attainment the health of the consumers. Being thus the objective of this work it was to evaluate and to guarantee the microbiological safety and the quality of foods produced in food unit through of the adapt of relative operational procedures to the hygienic cleaning. To make possible this estudy, observed, and verified preliminary stages of hygienic cleaning to the beginning of the work, after had been carried through awarenesses how much the methods, frequency and procedures of environment hygienic cleaning, equipment and utensils and hands. Realized verification and it collects of the samples of the equipment, utensils and hands used technique of "SWAB". The microbiological verifications seach been how much the mesophyllic aerobic microorganisms, mould and yeast, total coliform, termotolerants a 45°C coliform, positive Estafilococos coagulase, Salmonella spp, Pseudomonas aeruginosa.The results of the carried through analyses demonstrate significant reduction, in the microbiological requirements above of 99,5 % after accomplishment of training and awareness and the adoption of the procedures and instructions / A Organização Mundial de Saúde (OMS) indica que dentre as doenças de origem alimentar, mais de 60% dos casos decorrem de técnicas inadequadas de processamento envolvendo os microrganismos e parasitas patogênicos, além de seus produtos tóxicos. Quando se fala em gestão de qualidade na industrialização e manipulação de alimentos, é obrigatório lembrar dos Procedimentos Operacionais Padronizados(POP s), Boas Práticas de Fabricação (BPF s) e Análise de Perigos e Pontos Críticos de Controle (APPCC/HACCP). Em unidades de alimentação a qualidade e segurança estão relacionadas à produtividade. Para o aspecto de segurança do consumidor, estas ferramentas proporcionam influenciar conceitos de qualidade e segurança alimentar que necessitam ser fiscalizados e implementados conforme exigência da legislação. Para unidades de alimentação é relevante que estas também estejam comprometidas e sensibilizadas da importância em estabelecer procedimentos operacionais padronizados de higiene, com vistas às condições higiênicas sanitárias de equipamentos, utensílios e mãos de manipuladores. Proporcionando base técnica aos responsáveis e manipuladores sobre a obtenção de alimentos seguros a saúde dos consumidores. O objetivo deste trabalho foi avaliar e garantir a segurança microbiológica e a qualidade de alimentos produzidos em unidade de alimentação através da adequação de procedimentos operacionais relativos à higienização. Para viabilizar esse estudo, foram observadas e verificadas etapas de higienização preliminares ao inicio do trabalho, após foram realizadas conscientizações quanto a métodos, freqüência e procedimentos de higienização de equipamentos, utensílios e mãos. Realizou-se verificação e coleta das amostras dos equipamentos, utensílios e mãos de manipuladores usando técnica de SWAB . As verificações microbiológicas pesquisadas foram quanto a microrganismos aeróbicos mesofilos, bolores e leveduras, coliformes totais, coliformes termotolerantes a 45°C, Estafilococos coagulase positiva, Salmonella spp e Pseudomonas aeruginosa. Os resultados das análises realizadas demonstram redução significativa, nos requisitos microbiológicos, acima de 99,5% após realização de treinamentos e conscientização e a adoção dos procedimentos e instruções