Dissertations / Theses on the topic 'External coil'

In industrial drives market, speed and position estimation are one of the most important subjects for accurate motor drives. Vector controlled drives has the best dynamic performance among AC motor drives. Sensorless vector control is one of the most studied one. However, sensorless drive systems fail at low or zero speeds and may not have enough accuracy. For better accuracy and speed range speed sensors or position encoders are usually essential. However, coupling of sensor and sensor prices introduces extra cost on the drive. Thus in order to reduce the cost of the drive a cheap and easy to mount speed sensor is essential. Throughout this study, a speed and position sensor using an external search coil placed between cooling fins on the frame of an induction machine is proposed. The search coil utilizes the fringing flux outside the frame of induction motor. Using the induced voltage on the external search coil, a new method that estimates the flux and rotor position is proposed. In this study, the induced voltage on the search coils are investigated with different types of search coils placed on various positions. The frequency domain and time domain analysis are performed in order to build a model that can estimate machine flux, rotor speed and rotor position. As a result of this study, a low cost, easy to mount speed and position sensor is designed and implemented. Experiment results are presented.

<p> Although 1.5 and 3 Tesla (T) magnetic resonance (MR) systems remain the clinical standard, the number of 7 T MR systems has increased over the past decade because of the promise of higher signal-to-noise ratio (SNR), which can translate to images with higher resolution, improved image quality and faster acquisition times. However, there are a number of technical challenges that have prevented exploiting the full potential of ultra-high field (&ge; 7 T) MR imaging (MRI), such as the inhomogeneous distribution of the radiofrequency (RF) electromagnetic field and specific energy absorption rate (SAR), which can compromise image quality and patient safety. </p><p> To better understand the origin of these issues, we first investigated the dependence of the spatial distribution of the magnetic field associated with a surface RF coil on the operating frequency and electrical properties of the sample. Our results demonstrated that the asymmetries between the transmit (<i>B</i>₁⁺) and receive (<i>B</i>₁^&ndash;) circularly polarized components of the magnetic field, which are in part responsible for RF inhomogeneity, depend on the electric conductivity of the sample. On the other hand, when sample conductivity is low, a high relative permittivity can result in an inhomogeneous RF field distribution, due to significant constructive and destructive interference patterns between forward and reflected propagating magnetic field within the sample. </p><p> We then investigated the use of high permittivity materials (HPMs) as a method to alter the field distribution and improve transmit and receive coil performance in MRI. We showed that HPM placed at a distance from an RF loop coil can passively shape the field within the sample. Our results showed improvement in transmit and receive sensitivity overlap, extension of coil field-of-view, and enhancement in transmit/receive efficiency. We demonstrated the utility of this concept by employing HPM to improve performance of an existing commercial head coil for the inferior regions of the brain, where the specific coil&rsquo;s imaging efficiency was inherently poor. Results showed a gain in SNR, while the maximum local and head SAR values remained below the prescribed limits. We showed that increasing coil performance with HPM could improve detection of functional MR activation during a motor-based task for whole brain fMRI. </p><p> Finally, to gain an intuitive understanding of how HPM improves coil performance, we investigated how HPM separately affects signal and noise sensitivity to improve SNR. For this purpose, we employed a theoretical model based on dyadic Green&rsquo;s functions to compare the characteristics of current patterns, i.e. the optimal spatial distribution of coil conductors, that would either maximize SNR (ideal current patterns), maximize signal reception (signal-only optimal current patterns), or minimize sample noise (dark mode current patterns). Our results demonstrated that the presence of a lossless HPM changed the relative balance of signal-only optimal and dark mode current patterns. For a given relative permittivity, increasing the thickness of the HPM altered the magnitude of the currents required to optimize signal sensitivity at the voxel of interest as well as decreased the net electric field in the sample, which is associated, via reciprocity, to the noise received from the sample. Our results also suggested that signal-only current patterns could be used to identify HPM configurations that lead to high SNR gain for RF coil arrays. We anticipate that physical insights from this work could be utilized to build the next generation of high performing RF coils integrated with HPM.</p><p>

Corporate storytelling is a recognized marketing strategy, however the usage of it is regarded to be unclear from a theoretical perspective. Further, the internal usage of storytelling among organizations is more common than the external use. Hence, this research was aimed towards storytelling for external stakeholders.

Les progrès de la microfluidique, de la technologie sensorielle et de la biologie synthétique et moléculaire ont permis l'émergence d'un nouveau domaine scientifique dans lequel les principes fondamentaux de la théorie du contrôle peuvent être appliqués pour contrôler et réguler de manière externe les bioprocessus cellulaires : la cybernétique. Jusqu'à présent, la cybernétique a été capable de contrôler avec succès des réseaux génétiques complexes, multi-stables et adaptatifs au niveau de la population et de la cellule unique, mais les défis du contrôle de systèmes biologiques de type multi-agent composés de multiples composants interactifs n'ont pas encore été relevés. Dans cette étude, nous nous sommes concentrés sur des colonies denses d'E. coli, semblables à des biofilms, qui ont été cultivées à l'intérieur d'un dispositif microfluidique multicouche dont la géométrie permet la croissance des colonies dans une seule direction. De même que pour les biofilms, il est largement connu que les colonies denses d'E.coli présentent des niveaux remarquables d'organisation spatiale qui sont la conséquence de l'interaction complexe entre les gradients nutritifs et chimiques et les interactions métaboliques entre les différentes couches de la colonie. Ces interactions rendent les colonies denses et les biofilms plus résistants aux traitements antimicrobiens, ce qui les rend difficiles à éradiquer. La question de savoir si et dans quelle mesure nous pouvons contrôler ce système de l'extérieur reste ouverte. Pour répondre à cette question, nous avons d'abord analysé quantitativement les modèles de croissance à l'intérieur de la colonie pour comprendre la dynamique du système. Nous avons utilisé trois stratégies différentes pour perturber la colonie et voir l'impact sur les modèles de croissance spatiale - la modulation de l'ARN polymérase par un promoteur inductible et la modulation biochimique des ressources cellulaires par des changements de nutriments et des antibiotiques. Comme les cellules ne sont pas mobiles, la vitesse d'invasion de la colonie peut être considérée comme un descripteur global de la dynamique de croissance spatiale de la colonie. En gardant cela à l'esprit, nous avons finalement utilisé la compréhension de la dynamique des systèmes, la connaissance de la réponse des colonies à divers stimuli et une plateforme de contrôle faite sur mesure pour contrôler de manière externe la vitesse d'invasion de la colonie<br>Advances in microfluidics, sensory technology and synthetic and molecular biology enabled the rise of a novel scientific field in which fundamentals of control theory can be applied to externally control and regulate cellular bioprocesses-cybergenetics. So far, cybergenetics was able to successfully control complex multi-stable and adaptive gene networks at the population and the single cell level, but challenges in the control of biological multiagent-like system composed of multiple interactive components have not yet been addressed. In this study we focused on dense biofilm like colonies of E.coli which were grown inside the multilayered microfluidic device whose geometry enabled the growth of the colonies in one direction. Similarly to biofilms, it is widely known that dense E.coli colonies exhibit remarkable levels of spatial organization that come as a consequence of the complex interplay between nutrient and chemical gradients and metabolic interactions between different layers of the colony. These interactions make both dense colonies and biofilms more resistant to antimicrobial agents treatment consequently making them difficult to eradicate. Thus can we and at which extent we could externally control this system remains an open question. To answer this we firstly quantitively analyzed the growth patterns inside the colony to understand the dynamics of the system. We used three different strategies to perturb the colony and to see the impact on the spatial growth patterns - modulation of RNA polymerase by inducible promoter and biochemical modulation of the cellular resources by nutrient change and antibiotics. Since the cells were nonmotile, the invasion speed of the colony could be regarded as a global descriptor of the colony spatial growth dynamics. Thus having this in mind we finally used the understanding of the systems dynamics, knowledge of colonies response to various stimulus and a custom made control platform to externally control the invasion speed of the colony

This thesis focuses on the process of identity construction taking place within the European Union. Through an analysis of how the Union constructs the internal dimension of its identity in its domestic sphere targeting the citizens of its Member States and how it constructs the external dimension of its identity in the international sphere targeting mainly the non-member states and their citizens, the thesis seeks to answer the question to what extent the internal and external dimensions of the EU's identity, as analysed within the framework of the education policy and the foreign policy of the Union, form a coherent whole. It argues that identity has two dimensions: an external dimension which corresponds to the projection of the political community onto the world, and an internal one in which the idea of this imagined political community is projected back onto its citizens. Building on this framework, the thesis claims that these two facets of identity form a coherent and meaningful identity framework, the internal and external dimensions of which are constructed in quite similar and complementary ways. It also argues that the representations of the internal and external facets of the EU's identity illustrate great similarities in the way they were conceptualised, promoted and linked to the overall policy objectives of the Union in the two policy areas discussed within the framework of this study.

Nutrient runoff from excess land application of poultry litter in the Chesapeake Bay Watershed has caused damage to the Chesapeake Bay and lead to the need for alternative poultry litter disposal methods. This study provided an economic feasibility analysis of the use of poultry litter as a partial replacement of coal at an electrical generating unit in Virginiaâ s Chesapeake Bay Watershed. Previous research on the feasibility of converting litter to energy failed to include uncertainty in benefit-cost variables, therefore, this study used risk analysis to incorporate variable uncertainty. Project net worth in previous studies was measured under a public investment scenario with risk neutral preferences but did not take into account risk averse preferences common in private investment. This paper compared benefits under both public risk neutral and private risk averse investor preferences. NPV results showed the proposed project to be feasible but sensitive to the acquisition cost of poultry litter, the unit ash value of litter, and future coal price projections. The maximum level of risk aversion required for feasibility increased when expected returns were measured on an investment scale compared to an annual income scale. Poultry litter combustion produced lower levels of NOx and SO2 emissions compared to coal, therefore, emission allowance trading through the EPA market based trading programs generated additional benefits to the model and increased the maximum level of risk aversion permitted for feasibility. Results suggested the potential to dispose of 110 thousand tons of poultry litter per year from the Chesapeake Bay Watershed without violating EPA emission standards.<br>Master of Science

La membrane externe des bactéries à Qram négatif est composée de deux grandes classes de protéines. Les protéines en forme de tonneau bêta sont chaperonnées dans le périplasme par les protéines SurA, Skp et DegP et adressées à la membrane externe où elles sont insérées par le complexe Bam (beta-barrel assembly machinery). Les lipoprotéines (protéines ancrées dans la bicouche lipidique par trois acides gras) sont acheminées par la machinerie Loi (localisation of outer membrane lipoprotein), dont LolA, la navette périplasmique transmet la lipoprotéine au récepteur membranaire LolB, responsable de l'insertion des acides gras dans la membrane. La sécrétine PulD du système de sécrétion de type II de Klebsiella oxytoca, un multimère de 12 sous-unités et de SOOkDa, fait partie d'une autre famille, celle des protéines les plus proéminentes de la membrane externe. Afin de comprendre le mécanisme d'adressage de PulD dans le périplasme et au niveau de la membrane externe, nous avons dans un premier testé le rôle de la voie générale d'assemblage. Nous avons montré que le processus de multimérisation et d'insertion de PulD dans la membrane externe est indépendant de la machinerie Bam. L'implication des protéines chaperons générales dans le périplasme serait mineure mais reste à définir plus précisément. Dans un deuxième temps, nous avons étudié la relation de PulD avec sa lipoprotéine chaperon spécifique, PulS. Nous avons pour la première fois mis en évidence l'adressage d'une protéine non lipidée et intégrale de la membrane externe par la voie Loi, en identifiant un complexe périplasmique tripartite formé de LolA, d'une lipoprotéine chaperon spécifique, PulS (ou pilotine) et de PulD<br>The outer membrane of Gram negative bacteria is composed of two main protein classes. Beta-barrel proteins are chaperoned in the periplasm by SurA, Skp and DegP and adressed to the outer membrane where their insertion is dependent on the Bam complex (beta-barrel assembly machinery). Lipoproteins (proteins anchored in the lipid bilayer via three fatty acids) are targeted via the Loi machinery (localisation of outer membrane lipoprotein). The periplasmic shuttle LolA transfers the lipoprotein to the outer membrane receptor LolB, responsible for the fatty acid insertion in the membrane. The secretin PulD, a dodecamer complex of SOOKDa, of the Klebsiella oxytoca type II secretion System, is part of another class of protein: the largest proteins of the outer membrane. In order to understand the targeting and assembly mecanism PulD in the periplasm and at the outer membrane, we first investigated thé général assembly pathway. We showed that multimerisation and the insertion of PulD in the outer membrane is independent of the Bam machinery. The minor role of the general chaperons in the periplasm remain to be further described. Then, we studied the relation between PulD and its specific chaperon lipoprotein, PulS. We could highlight for the first time the targeting of an integral, non-lipidated outer membrane protein by the Loi machinery by identifying a periplasmic complex formed by LolA, PulS (or pilotin) and PulD

Made available in DSpace on 2014-06-11T19:25:18Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-15Bitstream added on 2014-06-13T20:13:35Z : No. of bitstreams: 1 lavagnini_tc_me_jabo.pdf: 549020 bytes, checksum: e38c026a1cc6fc8bb5122d61b6349138 (MD5)<br>Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)<br>Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)<br>Os crisopídeos são insetos com grande potencial para uso em programas de controle biológico de pragas agrícolas. Populações de Chrysoperla externa apresentam ampla distribuição geográfica, abrangendo desde o sul dos Estados Unidos até o sul da América do Sul, ocorrendo em diferentes ambientes. Contudo, há relativamente poucos estudos buscando compreender a estrutura genética de agentes de controle biológico, especialmente insetos predadores. Desta forma, os principais objetivos deste trabalho foram caracterizar geneticamente as populações de C. externa por meio de sequências do gene mitocondrial COI e compreender sua estrutura populacional nos municípios amostrados no Estado de São Paulo. Para tanto, indivíduos adultos foram coletados em pomares de citros, e da região torácica foi extraído o DNA total. O gene COI foi amplificado por meio da técnica de PCR e as amostras foram purificadas e sequenciadas. As populações de C. externa analisadas apresentaram elevada diversidade genética, bem distribuída entre os municípios amostrados. Esta homogeneização pode ser decorrência de fluxo gênico, ação antrópica, correntes de ar, proximidade das fazendas com matas nativas e elevado potencial reprodutivo de C. externa. A partir dos resultados obtidos é possível inferir que o agroecossistema, por ser um ambiente homogêneo, esteja contribuindo para a perda de estruturação que havia entre estas populações quando elas viviam em ecossistemas nativos, sendo assim, é fundamental que as populações de C. externa sejam estudas neste ambiente, para que possam ser compreendidas em seu habitat de origem e sem a influência da ação antrópica<br>The green lacewings are insects with great potential for use in programs of biological control of agricultural pests. Populations of Chrysoperla externa are widely distributed geographically, being found from the southern United States until the southern South America, occurring in different environments. However, there are relatively few studies trying to understand the genetic structure of biological control agents, especially predatory insects. Thus, the main objectives of this work were to characterize genetically the populations of C. externa through COI mitochondrial gene sequences and to understand its population structure in sampled municipalities in the State of São Paulo. For this purpose, adult individuals were collected in citrus orchards, and from its torax were extracted the total DNA. The COI gene was amplified by PCR and the samples were purified and sequenced. The populations showed high genetic diversity, well distributed among the municipalities. This homogenization may be due to gene flow, human action, action of winds, proximity of farms to native forests, and high reproductive potential of C. externa. From the results, is possible to infer that the agroecosystem, a homogeneous environment, may be contributing to the loss of structure that existed among the populations when they lived in native ecosystems, and therefore, the populations of C. externa must be studied in this environment, so they could be understood in its natural habitat and without the influence of the human action

As vacinas antimeningocócicas têm se demonstrado efetivas contra os sorogrupos A e C, no entanto ainda não existe vacina contra o sorogrupo B devido à similaridade entre a estrutura capsular do polissacáride B e o ácido polisiálico que faz parte do tecido cerebral humano, podendo levar à autoimunidade. O objetivo deste estudo foi investigar as propriedades adjuvantes dos toxóides Stx1 e Stx2 (STEC) de Escherichia coli, administrados em preparações antigênicas com vesículas de membrana externa nativa (NOMV) de Neisseria meningitidis B, comparando duas vias de imunização prime-boost ou intramuscular, em camundongos BALB/c com idade entre 6-8 semanas. A determinação dos níveis de anticorpos empregando a técnica de ELISA, mostrou elevadas concentrações de anticorpos IgG em soros de animais imunizados pela via intramuscular com Stx1+NOMV, mas não com NOMV, o que sugere que por esta via (intramuscular apenas) Stx1 possa ter atuado como adjuvante. No ensaio de Immunoblotting, soros de animais imunizados com Stx1+NOMV reconheceram maior número de antígenos de NOMV quando comparado ao grupo que recebeu Stx2+NOMV. O sistema prime-boost mostrou-se efetivo quando comparamos os níveis de anticorpos presentes no soro após a dose intramuscular (reforço), entretanto, não melhor do que quando utilizamos duas doses apenas pela via intramuscular. Este estudo poderá contribuir no desenvolvimento de tecnologias associadas a novas preparações antigênicas utilizando antígenos de membrana externa de N. meningitidis B , empregando toxóides como adjuvantes.<br>The meningococcal vaccines have been shown to be effective against serogroups A and C, however there is still no vaccine against serogroup B. The capsular polysaccharide from serogroup B meningococci polysialic acid moiety mimetic of many human glycoproteins including the neural cell adhesion molecules and may lead to autoimmunity. This study aimed to investigate the adjuvant properties of toxoids Stx1 and Stx2 (STEC) from Escherichia coli and native outer membrane vesicles (NOMV) of Neisseria meningitidis B, comparing two ways of immunization prime-boost or only intramuscular in BALB/c mice. The results showed high concentrations of IgG antibodies in sera of animals immunized intramuscularly with Stx1+NOMV, suggesting that in this way may have Stx1 acted as an adjuvant. In the Immunoblotting assay, sera from animals immunized with Stx1+NOMV recognized more antigens of NOMV when compared to the group that received Stx2+NOMV. The prime-boost was effective however, no better than only two doses intramuscularly. This study may contribute to the development of new technologies and strategies against N. meningitidis B employing toxoids as adjuvants.

Ag43 est une protéine de la membrane externe permettant à E. Coli d'auto-agréger. Elle promeut la formation de biofilms et est impliquée dans la persistance de souches d' E. Coli uropathogènes dans le tractus urinaire. Cette adhésine est régulée par un mécanisme de variation de phase très bien décrit : l'expression d'agn43 résulte de la compétition entre la méthylase Dam (activateur) et du régulateur transcriptionnel OxyR (represseur). C'est pourquoi des cellules clonales peuvent soit exprimer agn43 (phase ON) ou non (phase OFF). A l'heure de l'écriture de cette thèse, ce mécanisme reste le seul connu pour la régulation d'agn43, malgré que le rôle de la variation de phase in vivo soit toujours à l'étude. Bien que le 'switch' de phases d'agn43 conduit à une population hétérogène de bactéries ON et OFF, les études menées sur Ag43 considèrent rarement les biais potentiels associés à la variation de phase. Nous avons donc suivi le status ON / OFF d'agn43 génétiquement et phénotypiquement, et nous montrons que l'utilisation de populations ayant aléatoirement un status ON ou OFF pour agn43 peut entraîner des conclusions erronées sur la fonction ou de la régulation d'Ag43. En particulier, nous démontrons que Lrp et MqsR , précédemment identifiés comme des régulateurs d'agn43, ne régulent pas l'expression ou fréquence de commutation ON / OFF d'agn43. Nous montrons également que la formation de biofilms en conditions de flux dynamique n'influence pas la commutation ON / OFF, mais sélectionne physiquement les cellules agrégeant grâce à Ag43. Cela indique qu'une mauvaise interprétation est possible lorsque l'on étudie l'expression de tels gènes au sein de biofilms. De plus, nous apportons la preuve qu'ignorer l'état agn43 ON / OFF initial d'une population d'E. Con donnée risque de biaiser l'analyse des phénotypes associés à d'autres adhésines d'E. Coli, soulignant ainsi l'importance du suivi de la variation de phase d'Ag43. D'autre part, nous montrons que le système à deux composants CpxAR (ou Cpx), impliqué dans la réponse aux stress périplasmiques, peut moduler la fonction d'auto'-agrégation médiée par Ag43. Nous avons déterminé que le système Cpx ne régule pas directement l'expression d'Ag43 , ni sa stabilité, ni sa translocation à la surface mais plutôt indirectement par un mécanisme de « masquage / démasquage » via l'expression de firnbriae de type 1 (opéronfim), une autre adhésine majeure d'E. Coli également régulée par un mécanisme de variation de phase. De plus, nous avons montré que l'expression defim induit le système Cpx via la lipoprotéine N1pE, un inducteur du système Cpx, qui est impliquée dans la détection des surfaces, fournissant la première preuve d' une rétroaction négative des fimbriae de type 1 sur leur propre expression. En outre, nos résultats tendent à montrer qu'OxyR est aussi un répresseur de l'expression defim faisant de lui un régulateur clé des adhésines 'phase-variablez' d'E. Coli (Ag43 et Fim). Cette étude fournit donc un nouvel aperçu de la régulation d'Ag43 en particulier en ce qui concerne ses interactions avec d'autres adhésines<br>Ag43 is an outer-membrane protein enabling E. Coli to auto-aggregate. It promotes the formation of biofilms and is implicated in the persistence of uropathogenic E. Coli strains in the urinary tract. This adhesin is regulated by a mechanism of phase variation which is very well-documented : the expression of agn43 results of the competition between the Dam methylase (activator) and the transcriptional regulator OxyR (repressor). Fiente, sister-cells can either express agn43 (phase ON) or not (phase OFF). Up-to-date, this is the only known mechanism of regulation of agn43, although the in vivo role of phase variation is still poorly understood. Although the agn43 regulatory switch leads to a heterogeneous population of ON and OFF bacteria, studies of Ag43 seldom consider potential biases associated with phase variation. We monitored agn43 ON/OFF phase-variation status genetically and phenotypically and we show that the use of populations with random agn43 ON or OFF status could result in misleading conclusions about Ag43 function or regulation. In particular, we demonstrate that Lrp and MqsR, previously identified as agn43 regulators, do not regulate agn43 expression or ON/OFF switch frequency. We also show that biofilm formation in dynamic flow conditions does not influence agn43 ON/OFF switching but physically selects aggregating agn43 ON cells. This indicates that misinterpretation is possible when studying gene expression within biofilms. Moreover, we provide evidence that ignoring the initial agn43 ON/OFF status of the E. Coli populations studied is likely to bias analyses of phenotypes associated with other E. Coli adhesins thereby emphasizing the importance of monitoring Ag43 phase-variation. On another hand, we show that the two-component system CpxAR (or Cpx), implicated in periplasmic stress response, can modulate the function of Ag43- mediated auto-aggregation. We have determined that the Cpx system is not directly regulating Ag43 expression, stability nor its translocation to the surface but rather indirectly by a `masking/unmasking' mechanism via the expression of type 1 fimbriae (fim operon), another major phase-variable adhesin of E. Coli. Moreover, we have shown that the expression offim induces the Cpx system via the lipoprotein N1pE, inducer of the Cpx system, which is involved in surface sensing, providing the first evidence of a negative feedback of type 1 fimbriae on their own expression. Furthermore, our results tend to show that OxyR is also a repressor offim expression making it a master regulator of the phase-variable adhesins of E. Coli (Ag43 and Fim). This study therefore provides a new insight into Ag43 regulation in particular regarding its interactions with other adhesins

Made available in DSpace on 2014-06-11T19:25:19Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-02-24Bitstream added on 2014-06-13T18:26:17Z : No. of bitstreams: 1 baggio_mv_me_jabo.pdf: 512063 bytes, checksum: 11244a431e6c0f8cc5195b1c7d20e1bd (MD5)<br>Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)<br>Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)<br>Chrysoperla externa é uma espécie de crisopídeo encontrada em diversos agroecossistemas brasileiros, capaz de se alimentar de diferentes pragas agrícolas. Em cada ambiente em que for encontrada poderá sofrer diferentes pressões seletivas do ambiente. Para a verificação das mutações genéticas presentes nas populações de C. externa podem ser usados marcadores moleculares, em especial os genes mitocondriais, que são de fácil manipulação. C. externa é de fácil criação e a mais estudada para multiplicação massal, por isso se faz necessário estudar a resposta das populações quando liberadas em ambientes diferentes, visto que estas podem não sobreviver em áreas biogeográficas distintas, inviabilizando o seu papel como agente de controle de pragas. O objetivo deste trabalho foi caracterizar geneticamente as populações de C. externa nos municípios de Brotas (SP), Jaboticabal (SP), Rifaina (SP), São Carlos (SP), São José dos Campos (SP) e São Sebastião do Paraíso (MG). Para o gene COI foram verificados oito haplótipos, seis mutações e a maior diversidade haplotípica foi encontrada em Brotas e São Sebastião do Paraíso. Para o gene 16S foram observadas quatro mutações, seis haplótipos e a maior diversidade haplotípica ocorreu no município de São Sebastião do Paraíso. A distância genética encontrada entre as populações de C. externa não foi significativa para os dois genes analisados, evidenciando que provavelmente as populações são geneticamente compatíveis. O estudo da estrutura genética dessas populações de C. externa, de ambos os genes, mostrou que essas populações não apresentam um padrão de distribuição haplotípica. Então, talvez sejam necessários outros estudos com populações desta espécie oriundas de localidades mais distantes geograficamente dos que as utilizadas neste trabalho<br>Chrysoperla externa is a species of green lacewing found in several Brazilian agroecosystems able to feed on various agricultural pests. In each environment that is found may experience different environmental selective pressures. In order to verify genetic mutations in C. externa populations it may be used molecular markers, in particular mitochondrial genes, which are easy handling and extraction. C. externa is easy to create and the most studied for mass multiplication, so it is necessary to study on the response of populations when released in different environments, since they may not survive in different biogeographic areas, derailing its role as a pest controller. The objective of this study was to characterize genetically the populations of C. externa from the cities of Brotas (SP), Jaboticabal (SP), Rifaina (SP), São Carlos (SP), São José dos Campos (SP) and São Sebastião do Paraíso (MG). For the COI gene it was found eight haplotypes, six mutations and greater haplotype diversity in Brotas and São Sebastião do Paraíso. In the 16S there were four mutations, six haplotypes and haplotype diversity was higher in São Sebastião do Paraíso. The genetic distance among populations of C. externa was not significant for the two genes analyzed, showing that the populations are probably genetically compatible. The study of the genetic structure of populations of C. externa, showed for both genes that these populations do not show a pattern of haplotype distribution. So, it may need further study on populations of this species originating from geographically more distant locations of those used in this work

Pour acquerir le fer, rendu indisponible chez l'hote par suite de sa complexation aux transferrines, les bacteries pathogenes disposent de systemes de transport hautement specialises. L'expression de ces systemes est regulee par la concentration en fer, et se traduit par l'excretion d'un siderophore (compose chelateur du fer) et la synthese d'un recepteur proteique, specifique du siderophore ferrique, situe dans la membrane externe de la bacterie. Les deux principaux recepteurs de siderophores mis en evidence chez les escherichia coli septicemiques sont les proteines ivt a et fep a, qui correspondent respectivement aux siderophores aerobactine et enterobactine. Apres avoir montre que la presence, au niveau de la membrane externe, des proteines regulees par le fer accroit la protection induite par vaccination avec des corps bacteriens entiers, le but de ce travail a ete de produire les proteines ivt a et fep a, par recombinaison genetique, en absence de toute regulation par le fer. La comparaison des sequences nucleotidiques de trois genes ivta, isoles de souches e. Coli d'origines differentes, montre que la conservation antigenique de la proteine ivt a provient d'une stricte conservation du gene ivt a au sein de l'operon aerobactine. Les genes ivt a et fep a ont ete clones dans un vecteur d'expression. L'expression des genes se fait en l'absence de regulation par le fer et conduit a la synthese massive des proteines ivt a et fep a et de leurs precurseurs sous forme d'inclusions intracytoplasmiques, en meme temps que les recepteurs synthetises s'integrent de facon normale dans la membrane externe. Ces derniers sont fonctionnellement et antigeniquement identiques a leurs homologues naturels

Orientador: Sérgio de Freitas<br>Banca: Nilza Maria Martinelli<br>Banca: Karina Lucas da Silva Brandão<br>Resumo: Chrysoperla externa é uma espécie de crisopídeo encontrada em diversos agroecossistemas brasileiros, capaz de se alimentar de diferentes pragas agrícolas. Em cada ambiente em que for encontrada poderá sofrer diferentes pressões seletivas do ambiente. Para a verificação das mutações genéticas presentes nas populações de C. externa podem ser usados marcadores moleculares, em especial os genes mitocondriais, que são de fácil manipulação. C. externa é de fácil criação e a mais estudada para multiplicação massal, por isso se faz necessário estudar a resposta das populações quando liberadas em ambientes diferentes, visto que estas podem não sobreviver em áreas biogeográficas distintas, inviabilizando o seu papel como agente de controle de pragas. O objetivo deste trabalho foi caracterizar geneticamente as populações de C. externa nos municípios de Brotas (SP), Jaboticabal (SP), Rifaina (SP), São Carlos (SP), São José dos Campos (SP) e São Sebastião do Paraíso (MG). Para o gene COI foram verificados oito haplótipos, seis mutações e a maior diversidade haplotípica foi encontrada em Brotas e São Sebastião do Paraíso. Para o gene 16S foram observadas quatro mutações, seis haplótipos e a maior diversidade haplotípica ocorreu no município de São Sebastião do Paraíso. A distância genética encontrada entre as populações de C. externa não foi significativa para os dois genes analisados, evidenciando que provavelmente as populações são geneticamente compatíveis. O estudo da estrutura genética dessas populações de C. externa, de ambos os genes, mostrou que essas populações não apresentam um padrão de distribuição haplotípica. Então, talvez sejam necessários outros estudos com populações desta espécie oriundas de localidades mais distantes geograficamente dos que as utilizadas neste trabalho<br>Abstract: Chrysoperla externa is a species of green lacewing found in several Brazilian agroecosystems able to feed on various agricultural pests. In each environment that is found may experience different environmental selective pressures. In order to verify genetic mutations in C. externa populations it may be used molecular markers, in particular mitochondrial genes, which are easy handling and extraction. C. externa is easy to create and the most studied for mass multiplication, so it is necessary to study on the response of populations when released in different environments, since they may not survive in different biogeographic areas, derailing its role as a pest controller. The objective of this study was to characterize genetically the populations of C. externa from the cities of Brotas (SP), Jaboticabal (SP), Rifaina (SP), São Carlos (SP), São José dos Campos (SP) and São Sebastião do Paraíso (MG). For the COI gene it was found eight haplotypes, six mutations and greater haplotype diversity in Brotas and São Sebastião do Paraíso. In the 16S there were four mutations, six haplotypes and haplotype diversity was higher in São Sebastião do Paraíso. The genetic distance among populations of C. externa was not significant for the two genes analyzed, showing that the populations are probably genetically compatible. The study of the genetic structure of populations of C. externa, showed for both genes that these populations do not show a pattern of haplotype distribution. So, it may need further study on populations of this species originating from geographically more distant locations of those used in this work<br>Mestre

Oggetto principale del progetto di ricerca è stata la valutazione del comportamento energetico di componenti edilizi attraverso l’analisi sperimentale in situ con la realizzazione di mock up configurati per lo scopo. In prima battuta, lo studio si è concentrato sulle problematiche inerenti ai consumi energetici estivi, il comfort interno e la salubrità degli ambienti prefiggendosi l’obiettivo di analizzare nuove strategie per l’ottimizzazione dell’involucro edilizio. La tematica dell’isolamento termico è stata, quindi, declinata sotto diversi aspetti: tipologia dei materiali isolanti e le loro caratteristiche chimico-fisiche, tipologia delle strutture edilizie che necessitano di isolamento, principali parametri termici da considerare e infine la traduzione, in termini di sperimentazione in sito, di un modello comparativo tra involucri termici su una struttura esistente fortemente inerziale ma priva di isolamento termico: mock up realizzato a Fabriano. La necessità di raggiungere standard energetici sempre più stringenti si è generalmente tradotta con la scelta e l’utilizzo di materiali isolanti con alte prestazioni per coibentare le pareti dei nostri edifici. Una corretta valutazione dei vantaggi e degli svantaggi legati a questa tecnica costruttiva è della massima importanza al fine di comprendere al meglio il comportamento termico delle strutture opache. Pertanto, la ricerca portata avanti, riporta i dati sperimentali e analitici ottenuti dallo studio di una parete ad elevata inerzia termica coibentata con due diversi strati isolanti esterni (metodologia in gergo definita come isolamento “a cappotto”) per soddisfare i requisiti nZEB italiani sulla resistenza termica e focalizzandosi sulle problematiche nell’ambito del clima mediterraneo. Infatti, il surriscaldamento degli ambienti interni e, quindi, l'uso eccessivo dei sistemi di raffrescamento rappresenta uno dei principali problemi sia per la salute degli occupanti che per i consumi energetici, in particolar modo durante il periodo estivo. Ne consegue che, al fine di limitare questo problema, è necessaria una progettazione appropriata e/o un adeguamento energetico dell'involucro edilizio che utilizzi un approccio globale e sinergico. Altro argomento di ricerca, seguito parallelamente al primo, è stato lo studio dei cool materials da applicare in sistemi di raffrescamento passivo degli edifici declinando la tecnica del cool roof alle facciate degli edifici. L’obiettivo dello studio è stato quello di valutare gli effettivi benefici che i cool materials possono avere una volta applicati su pareti verticali, al posto delle tradizionali vernici per esterni, e valutarne la convenienza in termini di costi-benefici-durabilità. Questi materiali possono contribuisce alla riduzione della temperatura superficiale dell’agglomerato abitativo grazie ad un migliore bilancio energetico relativo alle superfici in gioco favorendo la mitigazione dell’effetto di isola di calore urbana. Inoltre, l’utilizzo di pareti con corrette stratigrafie e il giusto posizionamento dell’isolamento con materiali che rispondano a condizioni al contorno dinamiche, soprattutto nei climi Mediterranei come il nostro, possono rappresentare una risposta alle problematiche riguardanti il miglioramento dell’efficienza energetica del nostro patrimonio edilizio. Il progetto di ricerca sui cool materials ha previsto una fase preliminare di studio dei materiali, formulazione delle pitture sperimentali e, infine, la preparazione del mock up sul quale installare i pannelli da monitorare durante il periodo estivo. Riassumendo, la problematica della riqualificazione degli edifici rispetto all’isolamento estivo ha affrontato, principalmente, i seguenti argomenti: • l’inerzia termica e la sua relazione con l’isolamento termico dell’involucro nel periodo estivo; • lo studio dei materiali e la loro sperimentazione in condizioni reali, ovvero in regime dinamico; • l’applicazione sperimentale dei cool materials sulle pareti verticali esterne per valutarne l’efficacia in termini di contenimento delle temperature superficiali.<br>The main object of the research project was the evaluation of the energy behavior of construction components through in situ measurement and experimental analysis with dedicated mock-up. At first, the study has focused on issues related to energy consumption over the summertime, indoor comfort’s situations and environment’s salubrity with the aim of analysing new strategies towards building envelope’s upgrade. Therefore, the topic of thermal insulation has been listed into different aspects: the type of insulating materials and their chemical-physical characteristics, the type of building structures that require insulation, the main thermal parameters to be considered within the research and, at last, the translation, in terms of on-site test, of a comparative model between several thermal envelopes applied on a highly inertial structure lacking of thermal isolation: mock up made in Fabriano. The use of high-performance insulating materials to insulate the building walls is the necessary consequence to achieve the higher strict energy standards. The urgent need of reaching higher energetic standards has brought to choosing high-performance isolating materials to insulate buildings. A correct evaluation of pros and cons of this specific construction technique is extremely fundamental in order to achieve a better understanding of opaque structures thermal behaviour’s. Perhaps, the current research reports experimental and analytical data obtained from the study of a high thermal inertia surface insulated with two different external isolating layers, to meet the Italian nZEB requirements on thermal resistance and focusing on set of problems caused by the Mediterranean climate conditions. In fact, the indoor environment overheating and, consequently, the excessive use of cooling systems represents one of the main problems both for the occupants’ health and for energy consumption, especially during the summertime. It follows that, in order to decrease the problem, a global and synergistic approach is necessary to design energy efficient building envelopes. Another topic of the research, in line with the first one covered, has been conducted on studying cool materials to be applied to passive cooling techniques with the application of cool roof technique to the building facades. The aim of the study was to assess the benefits of those materials when applied to vertical envelopes instead of traditional outdoor coatings and evaluate their convenience in terms of costs-benefits-durability. These materials can contribute to exterior’s temperature reduction of the housing agglomeration thanks to a better energy balance of the surfaces, facilitating the mitigation of the urban heat island effect. Furthermore, resolutions to the problems concerning the energy improvement of our building heritage can be represented by the use of correct stratigraphy walls and the right placement of the insulating materials that respond to dynamic boundary conditions, especially in Mediterranean climate. The research project on cool materials involved a preliminary phase of the materials study, the formulation of the experimental paints and, at last, the mock up preparation on which to install the panels to be monitored over the summertime period. In summary, the problem of building energy upgrading in regard to summer insulation is focused on the main following topics: • thermal inertia and its relationship with the thermal insulation of the envelope over summertime period; • the study of materials and their experimentation in real conditions, or rather in a dynamic regime; • the experimental application of cool materials on external vertical walls in order to evaluate their beneficial impact on surface’s temperature.

L’adaptation au stress extra-cytoplasmique chez Escherichia coli dépend de l’activation du facteur essentiel sigmeE, normalement séquestré par la protéine trans-membranaire RseA. En réponse à un stress généré par la présence de protéines mal-formées dans le périplasme, sigmaE est relargué grâce au clivage successif de RseA par les protéases membranaires essentielles DegS et RseP. Nous avons isolé un suppresseur multicopie de la létalité observée en absence de RseP et DegS. Ce suppresseur code un nouveau ARN non-codant, RseX. Son activité et sa stabilité sont dépendantes de la protéine de liaison à l’ARN Hfq, avec laquelle il interagit in vitro. Afin d’identifier les ARNm cibles de RseX, nous avons développé une technique in vitro pour capturer ces cibles ARNm putatives. Les ARNm ompA et ompC, codant deux protéines majeures de la membrane externe, ont été identifiés. Une complémentarité de séquences entre RseX et la région d’initiation de la traduction des ARNm ompA et ompC avec a été mise en évidence. In vivo, les quantités des ARNm ompA et ompC et des protéines correspondantes sont diminuées dans une souche sur-exprimant RseX. Nous montrons RseP n’est plus essentielle dans le double mutant DompA DompC ; nous proposons que le phénotype de suppression de RseX envers l’absence de la protéase RseP s’explique par une perte de toxicité des protéines de la membrane externe OmpA et OmpC, rendant la voie d’adaptation au stress extra-cytoplasmique partiellement dispensable. Ce travail a ainsi contribué à révéler l’importance du contrôle de la quantité de protéines de la membrane externe par des ARN non-codant régulateurs dans le maintien de l’intégrité de l’enveloppe<br>In Escherichia coli, adaptation to extra-cytoplasmic stress depends on the activation of sigmaE, normally sequestered by the membrane protein RseA. SE is released in response to stress generated by accumulation of denatured proteins in periplasm through the successive RseA cleavage by DegS and the RIP protease RseP. SE and proteases that free it from RseA are essential. We isolated a multicopy suppressor that alleviated RseP and DegS requirement. The suppressor encodes a novel small non-coding RNA, RseX. Its activity and its stability require the RNA-binding protein Hfq. In order to identify mRNA RseX-targets, we developed an in vitro screen to capture RseX putative partners. OmpA and ompC mRNA, which encode two major outer membrane proteins, were identified. RseX activity was shown to confer an Hfq-dependent coordinate OmpA and OmpC down-regulation. We show that RseP is no longer essential in a strain lacking OmpA and OmpC; we conclude that the suppression phenotype is explained by the loss of toxicity of these two specific outer membrane proteins, giving the adaptation to extra-cytoplasmic stress signaling pathway partially dispensable. This work contributes to reveal the importance of quantity control of outer membrane proteins by regulatory small RNAs in the maintenance of envelope integrity

Las interacciones entre la microbiota y el huésped son complejas y dependen principalmente de factores secretados que pueden atravesar la capa de mucus y alcanzar el epitelio. Dentro de los factores secretados, las bacterias liberan vesículas extracelulares como mecanismo de comunicación con el entorno. Estas vesículas representan un mecanismo de secreción de proteínas y otros compuestos activos en un ambiente protegido que permiten la interacción a distancia con otras células. Puesto que Escherichia coli se encuentra formando parte de la microbiota normal del intestino humano, en este trabajo, se han utilizado como modelo de estudio las vesículas de membrana externa (OMVs) secretadas por la cepa probiótica E. coli Nissle 1917 (EcN) y la cepa comensal ECOR12. Como primer objetivo nos planteamos establecer la vía de internalización de las OMVs de EcN y ECOR12 en varias líneas celulares de epitelio intestinal. El análisis mediante espetrofluorimetría y microscopía confocal de fluorescencia de células incubadas con OMVs marcadas con rodamina B-R18 en presencia de inhibidores específicos de las vías de endocitosis ha permitido establecer que las OMVs de estas cepas son internalizadas en las células epiteliales mediante endocitosis mediada por clatrina. La colocalización de las OMVs con clatrina y marcadores específicos de endosomas (proteína EEA-1) y lisosomas (Lysotracker) demuestra el tráfico intracelular típico de esta vía, siendo por tanto dirigidas hacia compartimentos lisosómicos. También hemos demostrado que las OMVs de estas cepas no son citotóxicas puesto que no alteran la viabilidad celular si bien reducen la proliferación de células HT-29. El marcaje específico de aductos de 8-oxo-dG, reveló que, a diferencia de otras cepas de E. coli, las OMVs de EcN y ECOR12 no promueven este tipo de daño oxidativo. Sin embargo, el análisis por citometría de flujo y microscopía confocal de fluorescencia de la histona H2AX fosforilada (γH2AX) evidenció que OMVs de la cepa probiótica EcN producen una mayor cantidad de roturas de doble cadena en el DNA que las vesículas de la cepa comensal ECOR12. Las OMVs contienen múltiples componentes que actúan como ligandos de los receptores de reconocimiento de patrones microbianos presentes en la célula huésped. Uno de estos componentes es el peptidoglicano (PGN) reconocido específicamente por los receptores citoplasmáticos tipo NOD. Puesto que la entrada de las OMVs por endocitosis y su acceso a endosomas constituyen una de las vías de entrada del PGN al interior celular, nos planteamos analizar la implicación de los receptores NOD-1/NOD-2 en la señalización mediada por las OMVs de EcN y ECOR12. Para ello seguimos dos aproximaciones. Por una parte se analizó la respuesta inflamatoria inducida por las OMVs en células Caco-2 silenciadas con siRNA específicos para estos receptores. El análisis por RT-qPCR y ELISA de las citoquinas IL-6 e IL-8 confirmó la implicación de NOD-1, pero no de NOD-2 en la señalización mediada por OMVs. Por otra parte, demostramos mediante microscopía de fluorescencia la interacción de NOD- 1 con las OMVs, así como el reclutamiento de NOD-1 hacia los compartimentos endosomales en células epiteliales incubadas con OMVs. Se ha descrito que este paso es esencial para la activación de NOD-1 por su ligando específico. Uno de los mayores inconvenientes para los estudios con OMVs, es el bajo rendimiento el cual requiere una gran cantidad de cultivo inicial que dificulta el proceso. Para aumentar el rendimiento se ha descrito el uso de mutantes en proteínas de la envoltura celular que presentan un fenotipo de hipervesiculación. En este trabajo hemos obtenido OMVs a partir del mutante tolR derivado de la cepa EcN. Sin embargo, la aplicación de técnicas de TEM de alta resolución mostró una considerable heterogeneidad estructural en las muestras del mutante EcN tolR, que, además de las vesículas comunes, incluían otras estructuras atípicas. Los ensayos de internalización en células Caco-2 utilizando vesículas marcadas con rodamina B-R18 o DIO evidenciaron una menor tasa de internalización para las vesículas derivadas de EcN tolR, la cual correlacionaba con una menor capacidad de unión de las OMVs a la célula epitelial. Estos hallazgos que indican la heterogeneidad de OMVs del mutante tolR puede tener un impacto importante sobre la funcionalidad de las vesículas.<br>The microbiota and host interactions are complex and depend mainly on factors secreted that can pass through the mucus layer and reaching the epithelium. Factors secreted, the bacteria release extracellular vesicles as a mechanism for communication with the environment. These vesicles represent a mechanism of secretion of proteins and other active compounds in a protected environment that allow the distance interaction with other cells. Since Escherichia coli is forming part of the normal microbiota of the human intestine, in this work, have been used as study model (OMVs) secreted outer membrane vesicles by the probiotic strain e. coli Nissle 1917 (EcN) and the strain diner ECOR12. First objective we set ourselves to establish the internalization of the OMVs of NEC and ECOR12 road in several cell lines of intestinal epithelium. Analysis by spectro-fluorimetry and confocal microscopy fluorescence of cells incubated with OMVs marked with rhodamine B-R18 in the presence of specific inhibitors of Endocytosis pathways has helped establish that the OMVs of these strains are internalized in the epithelial cells through clathrin-mediated Endocytosis. The co-localization of the OMVs with clathrin and specific markers (EEA-1 protein) endosomes and lysosomes (Lysotracker) shows the typical intracellular trafficking of this pathway, being therefore directed towards lysosomic compartments. We have also shown that the OMVs of these strains are not cytotoxic since that does not alter cell viability while reducing the proliferation of HT-29 cells. The specific marking of 8-oxo-dG adducts, revealed that, unlike other strains of E. coli, the OMVs of EcN and ECOR12 do not promote this type of oxidative damage. However, the analysis by flow cytometry and microscopy, confocal fluorescence of histone H2AX phosphorylated (γH2AX) showed OMVs of the probiotic strain NEC to produce a greater amount of double-strand breaks in the DNA that the vesicles of the strain diner ECOR12. The OMVs contain multiple components that act as ligands of microbial pattern present in the host cell recognition. One of these components is the Peptidoglycan (PGN) specifically recognized by the cytoplasmic receptors type NOD. Since the entry of the OMVs by Endocytosis and their access to endosomes constitute one of the routes of entry of the PGN to the cell interior, we analyze the involvement of recipients NOD-NOD-1⁄2 in signaling mediated by the OMVs of NEC and ECOR12. For this purpose, we follow two approaches. On the one hand analyzed the inflammatory response induced by the OMVs in silenced Caco-2 cells with siRNA specific for these receptors. Analysis by RT- qPCR and cytokines IL-6 and IL-8 ELISA confirmed the involvement of NOD-1, but not NOD-2 signaling mediated by OMVs. On the other hand, we demonstrate using fluorescence microscopy NOD-1 with the OMVs interaction, as well as the recruitment of NOD-1 towards endosomals compartments in epithelial cells incubated with OMVS. He has been described that this step is essential for NOD-1 activation by its ligand specific. One of the biggest drawbacks for studies with OMVs, is the low performance which requires a large amount of initial culture which hinders the process. To increase performance, the use of mutants in the cell envelope proteins that exhibit a phenotype of hypervesiculation has been described. In this work we have obtained OMVs from the mutant tolR derived strain EcN. However, the application of high resolution TEM techniques showed considerable structural heterogeneity in the samples of the mutant EcN tolR, which, besides the common vesicles, included other atypical structures. Trials of internalization in Caco-2 cells using vesicles marked with rhodamine B-R18 or gave showed a lower rate of internalization vesicles derived from NEC for tolR, which correlated with a lower binding capacity of the OMVs to epithelial cell. These findings indicating the heterogeneity of the mutant tolR OMVS can have a significant impact on the functionality of the vesicles.

L'introduction de la these consiste en une revue sur differents systemes d'expression de peptides etrangers par fusion genetique in vitro a une proteine porteuse, en insistant sur l'immunogenicite des proteines hybrides. Les resultats experimentaux portent sur l'etude de lamb, une proteine de la membrane externe d'e. Coli k12 impliquee specifiquement dans l'adsorption de bacteriophages et dans le transport du maltose et des maltodextrines. Ils comportent deux aspects majeurs. D'une part, nous avons aborde l'etude de la topologie et des fonctions de lamb: par analyse de mutations conferant la resistance aux phages. Nous avons identifie plusieurs regions de la proteine impliquees specifiquement dans l'adsorption des phages et fait l'hypothese qu'elles etaient exposees en surface des bacteries. Nous avons montre que certaines de ces regions sont aussi impliquees dans le transport des sucres. Ces travaux nous ont permis de proposer un modele de repliement et d'organisation fonctionnelle de la proteine. Nous avons pu confirmer que plusieurs boucles de la proteine etaient exposees de part et d'autre de la bicouche lipidique, par insertion genetique in vitro en deux etapes d'un epitope etranger en differents sites de lamb. D'autre part, en utilisant lamb comme proteine pilote nous avons mis au point une methode genetique generale qui permet d'exprimer une grande variete de peptides etrangers en surface de bacteries gram#. Nous avons montre que l'insertion genetique d'epitopes viraux dans une boucle exposee de lamb permettait d'induire de fortes reponses anticorps contre ces peptides

Resumo: Os crisopídeos são insetos com grande potencial para uso em programas de controle biológico de pragas agrícolas. Populações de Chrysoperla externa apresentam ampla distribuição geográfica, abrangendo desde o sul dos Estados Unidos até o sul da América do Sul, ocorrendo em diferentes ambientes. Contudo, há relativamente poucos estudos buscando compreender a estrutura genética de agentes de controle biológico, especialmente insetos predadores. Desta forma, os principais objetivos deste trabalho foram caracterizar geneticamente as populações de C. externa por meio de sequências do gene mitocondrial COI e compreender sua estrutura populacional nos municípios amostrados no Estado de São Paulo. Para tanto, indivíduos adultos foram coletados em pomares de citros, e da região torácica foi extraído o DNA total. O gene COI foi amplificado por meio da técnica de PCR e as amostras foram purificadas e sequenciadas. As populações de C. externa analisadas apresentaram elevada diversidade genética, bem distribuída entre os municípios amostrados. Esta homogeneização pode ser decorrência de fluxo gênico, ação antrópica, correntes de ar, proximidade das fazendas com matas nativas e elevado potencial reprodutivo de C. externa. A partir dos resultados obtidos é possível inferir que o agroecossistema, por ser um ambiente homogêneo, esteja contribuindo para a perda de estruturação que havia entre estas populações quando elas viviam em ecossistemas nativos, sendo assim, é fundamental que as populações de C. externa sejam estudas neste ambiente, para que possam ser compreendidas em seu habitat de origem e sem a influência da ação antrópica<br>Abstract: The green lacewings are insects with great potential for use in programs of biological control of agricultural pests. Populations of Chrysoperla externa are widely distributed geographically, being found from the southern United States until the southern South America, occurring in different environments. However, there are relatively few studies trying to understand the genetic structure of biological control agents, especially predatory insects. Thus, the main objectives of this work were to characterize genetically the populations of C. externa through COI mitochondrial gene sequences and to understand its population structure in sampled municipalities in the State of São Paulo. For this purpose, adult individuals were collected in citrus orchards, and from its torax were extracted the total DNA. The COI gene was amplified by PCR and the samples were purified and sequenced. The populations showed high genetic diversity, well distributed among the municipalities. This homogenization may be due to gene flow, human action, action of winds, proximity of farms to native forests, and high reproductive potential of C. externa. From the results, is possible to infer that the agroecosystem, a homogeneous environment, may be contributing to the loss of structure that existed among the populations when they lived in native ecosystems, and therefore, the populations of C. externa must be studied in this environment, so they could be understood in its natural habitat and without the influence of the human action<br>Orientador: Sérgio de Freitas<br>Coorientador: Adriana Coletto Morales<br>Banca: Sergio Antonio de Bortoli<br>Banca: Fernando de Faria Franco<br>Mestre

N.meningitidis é diplococcus gram-negativo, patógeno estritamente humano que similarmente a outras bactérias é circundado por membrana externa, com lipídios, proteínas (OMP) e lipopolissacárides. Ela tem sido uma das principais causas da meningite e de outras infecções invasoras no mundo. Este trabalho buscou usar o toxóide STX2 de E.coli como adjuvante para um possível e futuro modelo vacinal e como estimulante antigênico, proteínas da membrana externa do meningococo (OMP) transportados em lipossomas. Observaram-se diferenças na produção de anticorpos IgG obtidas entre os camundongos após cada uma das 3 sangrias mas, não quanto ao índice de avidez. A nova preparação antigênica desencadeou um alto título, mesmo após um ano da 1ª imunização, estimulou a produção de anticorpos para outros sítios de ligação e serviu como proteção ao LPS residual dos processos com deoxicolato da OMP, diminuindo toxicidade da preparação IM reduzindo os riscos para idosos e crianças muito pequenas e também, em imunizações de longo termo, com grande vantagem aos sistemas tradicionais.<br>N.meningitidis is diplococcus gram-negative strict human patogen that similarly to other bacteria are surrounded by external membrane with lipids, proteins (OMP) and LPS. It has been one of the main causes of the meningitidis and other invading infections in the world. This work searched to use STX2 toxoid of E.coli as adjuvant for a possible and future vaccine model and as antigenic stimulant proteins of the external membrane of meningococci (OMP) carried in liposomes. Differences in the production of IgG antibodies gotten between the mice each one of the 3 bleedings had been observed after but not how much to the avidity index. The new antigenic preparation unchained one high heading exactly after one year of 1st immunization stimulated the production of antibodies for other sites of linking and served as protection to the residual LPS of the processes with deoxicolate of the OMP diminishing toxicity of IM preparation reducing the aged risks for and very small children e also, in immunizations of long term with great advantage to the traditional systems.

A proteína Nef do vírus da imunodeficiência humana tipo 1 (VIH-1) é uma proteína miristilada, de 27 kDa, expressada em níveis elevados imediatamente após a infecção. Embora seja dispensável para a replicação viral in vitro, Nef parece desempenhar um papel fundamental ao nível da patogénese viral in vivo. Entre as inúmeras funções biológicas que lhe são atribuídas, contam-se a estimulação da replicação viral, a indução do aumento da infecciosidade dos viriões, a modulação negativa da expressão superficial do receptor celular CD4 e do complexo principal de histocompatibilidade de classe I (MHC I), a modulação de inúmeras vias de sinalização celular/transdução de sinal em linfocitos T e a interferência no processo de indução da morte celular programada, protegendo da apoptose as células infectadas e induzindo-a nas células vizinhas não infectadas, incluindo linfocitos T CD8+ específicos para o VIH-1. Devido ao seu papel fundamental ao nível do ciclo replicativo viral, a proteína Nef foi considerada como um potencial alvo terapêutico e vacinal. Neste trabalho, o gene nef do VIH-1 foi amplificado por PCR a partir da linha celular 8E5/LAV (HIV-1) e clonado no sistema de expressão “transportador-adjuvante” pVUB3 (Cote-Sierra et al., 1998), baseado na lipoproteína OprI da membrana externa de Pseudomonas aeruginosa. Desta clonagem resultou a construção do plasmídio recombinante pVUB3nef8E5, de 4958 pb, a partir do qual se induziu a expressão da proteína de fusão OprI-Nef ao nível da membrana externa de Escherichia coli. A produção da proteína de fusão foi demonstrada por visualização de extractos proteicos de membrana externa em gel de poliacrilamida/SDS e confirmada por experiências de imunodetecção com anticorpos anti-OprI e anti-Nef. Com o objectivo de obter extractos da proteína de fusão com um grau de pureza elevado, um oligonucleótido 6xHis foi introduzido a jusante da construção híbrida oprI-nef de pVUB3nef8E5, no que resultou a formação do novo vector de expressão pVUB3nefB-6xHis (4944 pb). Após um processo de optimização das condições experimentais relativas à purificação, sob condições desnaturantes, da proteína de fusão OprI-Nef-6xHis por cromatografia de afinidade e um passo adicional de ultrafiltração, foi possível a obtenção de extractos proteicos finais com um grau de pureza relativamente elevado (análise em gel de poliacrilamida/SDS e por imunodetecção). Estes apresentaram uma concentração proteica aproximada de 4 μg/μl, correspondendo a um rendimento global do processo de purificação de cerca de 1,5 mg de proteína por litro de cultura bacteriana, e uma quantidade de lipopolissacáridos (endotoxinas) adequada à realização de experiências de imunização. A proteína de fusão OprI-Nef-6xHis foi utilizada como imunogénio em experiências de imunização em modelo murino, com o objectivo de se caracterizar a componente humoral da resposta imune anti-Nef, por comparação com a administração da proteína Nef em tampão PBS (rNef) e em adjuvante de Freund (rNef/AF). A imunização com a proteína de fusão induziu uma resposta humoral anti-Nef caracterizada por um título de diluição limite de anticorpos da classe IgG muito elevado (1:37300), significativamente superior ao obtido no grupo rNef, sem que a sua administração tivesse aparentemente qualquer tipo de efeitos secundários nos animais. Por outro lado, comparativamente aos grupos rNef e rNef/AF, observou-se um desvio da resposta imune no sentido Th1, facto relevante no caso da infecção pelo VIH. Este fenómeno, bem com a indução de um título elevado de anticorpos anti-Nef da classe IgG, dever-se-á, muito provavelmente, à presença da componente lipídica na proteína de fusão. Finalmente, a utilização da proteína de fusão OprI-Nef-6xHis como antigénio em experiências de ELISA permitiu detectar anticorpos anti-Nef em 77% dos soros testados de indivíduos infectados com o VIH, indicando que a proteína híbrida obtida, embora purificada de um modo desnaturante, manterá epitopos de Nef sob uma forma imunologicamente relevante. Os resultados obtidos apontam ainda para um reconhecimento imune alargado destes epitopos por anticorpos naturais anti-Nef produzidos contra diferentes genótipos do VIH-1, e, muito provavelmente, mesmo contra o VIH-2, o que parece substanciar a utilização desta proteína de fusão como potencial imunogénio anti-VIH. Em conclusão, o sistema de expressão pVUB3 parece constituir um modelo promissor para a produção heteróloga, em bactérias Gram-negativas, de lipoproteínas de fusão contendo determinantes antigénicos de proteínas dos vírus da imunodeficiência humana, a serem eventualmente incluídas como agentes de imunização em esquemas experimentais de vacinação anti-VIH.<br>The human immunodeficiency virus type 1 (HIV-1) Nef protein is a 27 kDa myristylated early protein, expressed immediately after infection at relatively high levels. Although not necessary for viral replication in vitro, it has been shown to be a major determinant for AIDS pathogenesis. Nef can exert several effects at the cellular level: enhancement of viral replication and virion infectivity, downregulation of the cell-surface expression of CD4 and MHC class I molecules, modulation of T cell signal transduction pathways and regulation of programmed cell death (protecting infected cells from apoptosis and leading to bystander cell killing, including of HIV-specific cytotoxic T lymphocytes). Therefore, Nef has been considered a valuable target for the development of novel antiviral therapies and/or vaccines. In this work, HIV-1 nef gene from 8E5/LAV (HIV-1) cell line was cloned into the carrier-adjuvant plasmid system pVUB3 (Cote-Sierra et al., 1998), based on the major lipoprotein (OprI) from the outer membrane of Pseudomonas aeruginosa. This resulted in the construction of the 4958-bp recombinant plasmid pVUB3nef8E5, which allowed the inducible production of an outer membrane-bound OprI-Nef fusion protein in Escherichia coli. The expression of OprI-Nef was firstly demonstrated by SDS-PAGE and further confirmed by Western blotting with anti-OprI and anti-Nef antibodies. In order to obtain high purity OprI-Nef extracts, a 6xHis tag was introduced downstream of oprI-nef in pVUB3nef8E5, which resulted in the construction of the expression vector pVUB3nefB-6xHis. The expression of the new inducible fusion protein (OprI-Nef-6xHis) was confirmed by SDS-PAGE and immunoblotting, being the protein purified under denaturing conditions by immobilised metal affinity chromatography, followed by ultrafiltration. The purified final extracts had a protein concentration of 4 μg/μl, corresponding to a global purification yield of 1,5 mg of protein/litre of bacterial culture, and low levels of endotoxins, adequate for immunization experiments. Aiming at the characterization of humoral immune responses induced by immunization with Nef as a fusion protein with OprI, three groups of mice were immunized, respectively, with OprI-Nef-6xHis, Nef in the presence of Freund’s adjuvant or Nef alone in PBS. Mice immunized with the fusion protein developed high levels of IgG anti-Nef antibodies (endpoint titre of 1:37,300), which was significantly higher than in mice immunized with the protein alone, with no visible adverse side effects for the inoculated animals. On the other hand, the analysis of the isotypic patterns of anti-Nef antibodies showed that immunization with Nef protein, with or without adjuvant, yielded a preponderance of IgG1 antibodies, indicating a predominant Th2 immune response, whereas OprI-Nef-6xHis immunization biased the humoral response towards IgG2a production, indicating the preferential induction of a Th1 immune response. As a whole, these results most probably reflect the in-built adjuvanticity and immune response modulation capacity of OprI-Nef-6xHis by virtue of its lipid moiety. Finally, an ELISA-based method for detection of anti-Nef antibodies in sera from HIV infected individuals was developed, using the fusion protein OprI-Nef-6xHis as coating antigen. On the overall, 77% of the tested sera were considered positive, irrespective of HIV-1 env genotypes and, probably, even of HIV type. This result shows that, despite of OprI-Nef-6xHis biochemical purification under denaturing conditions, there is a broad immune recognition of Nef epitopes in the fusion protein by natural anti-Nef antibodies, what seems to support its future use as an anti-HIV immunogen. In conclusion, the data obtained in this work indicate that pVUB3 constitutes an appropriate expression system for the heterologous production of bacterial fusion lipoproteins containing HIV antigenic determinants, to be potentially included in experimental anti-HIV immunization protocols.

L'objectif de ma these a ete l'identification et la caracterisation de nouveaux facteurs d'initiation de la transcription plastidiale, associes a l'arn polymerase pep et a l'activite transcriptionnelle nep2. Dans un premier chapitre, nous mettons en evidence la presence de six facteurs de type sigma chez a. Thaliana : les proteines sig. In vivo, nous montrons que le facteur sigma chimerique 7 0 / sig1 permet la complementation du mutant d'e. Coli cag1 (rpod). Nous montrons egalement que les proteines sig1, sig2 et sig3 presentent, in vitro, une affinite variable pour les promoteurs plastidiaux et pour l'enzyme coeur d'e. Coli. En conclusion, nous proposons un classement provisoire de sig1 dans le groupe 1 des facteurs sigma essentiels, et suggerons que les proteines sig peuvent etre considerees comme les commutateurs genetiques de l'enzyme pep plastidiale. Dans un deuxieme chapitre, nous avons identifie un nouveau facteur b : la proteine brp. Les facteurs brp definissent un nouvel embranchement des homologues a tfiib, specifique aux plantes. Nous avons montre que ces proteines sont ancrees a la membrane externe des enveloppes plastidiales. L'identification de cette nouvelle famille de facteurs b suggere la presence d'un quatrieme systeme transcriptionnel eucaryotique dans les plantes, dont le role est certainement relie aux fonctions plastidiales. Les implications fonctionnelles de cette decouverte sont discutees et deux modeles moleculaires sont proposes.

碩士<br>國立中興大學<br>化學工程學系所<br>107<br>Photosynthesis has contributed a lot to the recovery of global carbon dioxide, which requires a key enzyme, ribulose 1,5-diphosphate carboxylase/oxygenase (Rubisco). In our previous studies, we has used microbes to develop a carbon fixation platform, introducing part of the Calvin cycle, including phosphoribosyl kinase (PrkA) and Rubisco, which are co-expressed in Escherichia coli (E.coli). In order to enhance the carbon fixation effect, it is necessary to enhance the enzyme activity of Rubisco. Based on the stoichiometric calculation results, we decided to provide additional energy, Nicotinamide adenine dinucleotide (NADH) to improve ability of carbon fixation. Glycerol is selected as the best substrate because two moles of NADH can be obtained by consuming one mole of glycerol, which is more reductive than common sugars such as glucose and xylose. By supplying additional energy, the Rubisco system can be used as an electron sink and improve the carbon flow to the C-2 product for better carbon recovery. Taking fructose as an example, the metabolic yield of E. coli/Fru+B+Gly reached 2.2±0.12 mole/mole, which was close to the highest theoretical yield, which represented Rubisco contributing the most during the fermentation process. In addition, we heterogeneously introduced the plastid pLOI295, which promotes the conversion of pyruvate to ethanol through the pdc and adh genes, promotes the positive pressure of NADH, reducing the accumulation of acetate, stimulates the consumption of carbon sources and makes up for the demand of ATP. The final metabolic yield was increased to 2.4 ± 0.36 mol/mol, wherein the ethanol yield was greatly increased from 0.25 ± 0.05 to 1.72 ± 0.24.

碩士<br>中原大學<br>機械工程研究所<br>98<br>The purpose of this study is to use Externally Warped Coils Induction Heating to determine the practicality of its application on mold surface heating. The first step is a test of heating efficiency using parallel and series type coils. The second step is the selection of the better coil type for mold temperature homogeniety. The third step is testing different mold designs and observing their effect on heating efficiency. Finally, we compare and analyze the experimental results with ANSYS® analysis software. Experimental results show the heating efficiency of series type coils is 8.26oC/s and the efficiency of parallel type is 1.88oC/s. The series type heats more rapidly and is, thus, a better system for heating the mold. Monitoring the surface temperature homogeniety between the mold cavity and core, we find the cavity surface temperature at positions T1, T2, and T3 heated at a rate of 8.13oC/s, 8.97oC/s, and 10.16oC/s respectively. The core surface temperatures at the same positions T1, T2, and T3 rose at rates of 7.83oC/s, 8.95oC/s, and 9.94oC/s respectively. The temperatures at these three points show homogeniety and symmetry. For different coil systems, their heating efficiency is directly related to the number of coils. The five, six, and seven coil systems have a heating efficiency of 4.9oC/s, 8.26oC/s, and 10.58oC/s respectively. Thus, the seven coil system is more advantageous than the others. Different spacing between the mold core and mold cavity does not adversely affect heating efficiency and homogeniety. For different coil intervals, the 18mm, 20mm, and 22mm distances have heating efficiencies of 9.81oC/s, 8.26oC/s, and 7.04oC/s respectively. From this, it is evident that an 18mm coil interval is more advantageous for heating efficiency. Experimental results show different coil systems and coil intervals affect the heating efficiency of the mold surface whereas the interval between the mold core and mold cavity does not have an adverse effect. The comparison between ANSYS® simulation result and experiment result show the same trend for mold surface temperature. This experiment shows that ANSYS® can be used for simulation and give accurate result on the Externally Warped Coils Induction Heating process which generates an efficient and even heating effect on mold surface temperature fields.

碩士<br>國立清華大學<br>生物資訊與結構生物研究所<br>101<br>Synthetic biology aims both to employ standard interchangeable components to mimic biological behavior, and to construct novel artificial biological systems with prescribed functions. In this study, the GA-based searching method was used to efficiently select an adequate set of promoter-RBS components from the well-characterized promoter-RBS libraries, and robust externally tunable synthetic biological filter devices with a specified input/output (I/O) response were built in Escherichia coli. The thresholds of these gene circuits could be tuned by adjusting the concentration of the control inducer. The green fluorescent protein (GFP) was employed as the reporter protein in the gene circuit, of which expression levels were measured using an ELISA reader. The gene expression patterns of these biological filters exhibited low-pass and high-pass like characteristics respectively in response to an input chemical gradient in vivo. In the future, different promoter-RBS components with different kinetic activities may be selected to precisely tune the threshold of the I/O response of filters, thereby making the experimental results more closely fit the model prediction. The biological filters may provide a wide variety of applications in pattern formation, biosensor, and cancer therapy.

碩士<br>淡江大學<br>化學學系<br>87<br>2.Electron Capture Detection Mass Spectrometry is a sensitive and selective technique for the analysis of compounds containing electrophilic functional groups. It has been used extensively for the analysis of many classes of compounds in the traditional mass spectrometers but none was reported from the ion trap mass spectrometer since the traditional ion trap MS can not perform negative ion analysis. However, in this study we have examined a series of Phthalic anhydride and their derivatives by electron capture detection in the ion trap tandem mass spectrometers with an external source (the Finnigan GCQplus). The GCQ is the first commercial benchtop ion trap mass spectrometer with the capability to perform negative ion analysis. In this study, a series of halogenated Phthalic anhydride and their derivatives will be examined. Methane was used as the reagent gas. Since these halogenated compounds possess higher electron affinities, negative ions can be formed perdominantly through a resonance electron capture process. Besides, using the tandem in time ion trap mass spectrometry can select a specific m/z of ion to perform MS/MS. Then the selected precursor ions dissociate by the collisional activation technique. From the fragments of parent ion, the structure of ions can be determined. These noval experiments can provide valuable results for negative ions which could not be detected by an traditional internal ionization ion trap mass spectrometer in the past.