Tesis sobre el tema "PNA Probes"

The current environment for drug discovery and disease treatment relies heavily on genomic analysis, structural biology and chemical biology techniques. With the enormous advances in genomic analysis and structural biology, the use of and desire for targeted therapies has increased. However, as more genomic data for cancer disease state pathology becomes available we must ask increasingly difficult questions and even produce new technologies, such as activity-based probes, to answer these questions. In particular, targeted kinase inhibitors for the treatment of cancer has become a mainstay for drug development for both industry and academia, but it is evident that the genomic data is not always indicative of protein expression. Additionally, protein expression alone does not completely characterize functional activity. Therefore, in order to more accurately validate drug targets and predict drug efficacy, we must not only identify possible targets but also determine their activity in vivo. The goal of this work was to develop a probe for Protein Kinase A that would act by alkylating a conserved cysteine in the substrate-binding pocket of the enzyme. We hypothesized that by targeting the substrate-binding pocket we could effectively utilize the natural substrate selectivity filters as well as take into account multiple endogenous regulatory mechanisms. We produced probes utilizing portions of the pseudosubstrate inhibitor PKI that demonstrate the ability to label the catalytic subunit of Protein Kinase A in an activity-dependent manner, thus making it an important first step in a new class of activity-based probes for the kinome.

The Programmable Logic Array (PLA) has been widely used in the design of VLSI circuits and systems because of its regularity, flexibility, and simplicity. The equivalence problem is typically to verify that the final description of a circuit is functionally equivalent to its initial description. Verifying the functional equivalence of two descriptions is equivalent to proving their logical equivalence. This problem of pure logic is essential to circuit design. The most widely used technique to solve the problem is based on Binary Decision Diagram or BDD, proposed by Bryant in 1986. Unfortunately, BDD requires too much time and space to represent moderately large circuits for equivalence testing. We design and implement a new algorithm called the Cover-Merge Algorithm for the equivalence problem based on a divide-and-conquer strategy using the concept of cover and a derivational method. We prove that the algorithm is sound and complete. Because of the NP-completeness of the problem, we emphasize simplifications to reduce the search space or to avoid redundant computations. Simplification techniques are incorporated into the algorithm as an essential part to speed up the the derivation process. Two different sets of heuristics are developed for two opposite goals: one for the proof of equivalence and the other for its disproof. Experiments on a large scale of data have shown that big speed-ups can be achieved by prioritizing the heuristics and by choosing the most favorable one at each iteration of the Algorithm. Results are compared with those for BDD on standard benchmark problems as well as on random PLAs to perform an unbiased way of testing algorithms. It has been shown that the Cover-Merge Algorithm outperforms BDD in nearly all problem instances in terms of time and space. The algorithm has demonstrated fairly stabilized and practical performances especially for big PLAs under a wide range of conditions, while BDD shows poor performance because of its memory greedy representation scheme without adequate simplification.

This thesis dealt with the issues of the emergence of Pan-Turkism in Ottoman Empire and of Pan-Turanism in Hungary between the years 1890 and 1920. The theoretical discussion and literature review related to the subject exhibited that these two nationalisms were possible only when a state bureaucrats and intellectuals try to save the state from collapse and make discussions on the national issues, or when a state elites and noble classes aim to use national ideology for protecting the state from external threats and providing benefits on behalf of national interest. While former suits to Ottoman Pan-Turkism, latter describes Hungarian Pan-Turanism. The thesis consisted of three main and related parts. The first part focused on the historical and theoretical development of nationalism and pan movements, and condition of pan movement in the context of theories of nationalism. In the second part, the emergence of Turkish nationalism and Pan-Turkism was analysed in the historical context. The third part dwelt upon the genesis of Pan-Turanism in Hungary, and its relations with Ottoman Pan-Turkism until the end of the First World War. For that reason, firstly, historical roots of Turkish nationalism and Pan-Turkism were sought so that it is able to see how the attempts to modernization in the Ottoman state provided a ground for the spreading of Western political concepts and ideas and the emergence of a secular nationalist intelligentsia. Also the role of Turcology and the influence of Russian Turks on the development and politicization of Turkish nationalism and Pan-Turkism could be assessed. Secondly, the political condition of Hungary in the nineteenth century was exposed in order to explain the emergence and development of Pan-Turanism. Then, the focus was made on the linguistic debates of Hungarian academic circles on the origin of Hungarians. Exposing the political and cultural conditions could facilitate to project the partnership between Pan-Turkism and Pan-Turanism. Throughout the thesis, it was tried to be demonstrated that Ottoman Empire and Hungarian state had different political conditions and necessities. While Ottoman state bureaucrats and intellectuals aimed to save the state
Hungarian elites and intellectuals urged on the Hungarian national interests. Although some strong relations and partnerships were manifest in political and cultural areas, Hungarian Pan-Turanists and Ottoman Pan-Turkists belonged to different state traditions. Turkish nationalism and Pan-Turkism had an aim to save the state and create a new national identity. Nevertheless, Hungarian nationalism and Pan-Turanism tended towards the national interests of Hungarians through expansionist policy. That was the reason why the relations between Pan-Turkists and Pan-Turanists remained temporary.

Computational simulations of chemical systems play an ever-increasing role in many areas of biochemical research from rational drug design to probing fundamental physiological processes. Depending on the method, a vast array of properties are able to be predicted. Here we report the design and implementation of two methods for investigating diverse problems in protein biochemistry. In order to better understand protein–metal interactions—most importantly for the difficult to model transition metal ions— empirical force field parameters were developed for Pt(II), cisplatin, and other Pt(II) coordination compounds. Two force field frameworks were used: a modified version of the fixed- charge OPLS-AA and the polarizable POSSIM force field. A seven-site model was used for the Pt(II) ion. The produced parameters are compatible with the OPLS-AA and POSSIM force fields and can be used in protein–metal binding simulations in which—contrary to the common treatment of metal ions in such simulations—the position or even coordination of the ion does not have to be constrained using preexisting knowledge. It has been demonstrated that the produced models are capable of reproducing key properties of relevant Pt(II) complexes but that the POSSIM formalism yields more accurate values for energies of formation than the OPLS-AA model. This Pt(II) model was employed—along with previously developed Cu(I) parameters—to investigate the binding of platinum to the protein Atox1, a human copper chaperone implicated in the resistance mechanism of cisplatin and other platinum antitumor compounds. In collaboration with the Dmitriev and Bernholc groups, we used our models to inform and refine spectroscopic experiments as well as to serve as starting points for high-performance quantum calculations. It was shown that under physiological redox conditions, copper(I) and cisplatin can form large polymers with glutathione. These polymers were capable of transferring copper(I) to apo-Atox1 or to platinum(II) to copper-loaded Atox1. Analysis of the simultaneous binding of copper(I) and platinum(II) to Atox1 was found to occur through the formation of copper–sulfur–platinum bridges, where copper is coordinated by three sulfur atoms and platinum by four sulfur atoms. With the goal of using a simple model to be able to quickly estimate the acid disassociation constants of proteins, PKA17 has been developed and tested. PKA17 is a coarse-grain grid-based method and software tool for accurately and rapidly calculating protein pKa values given an input PDB structure file. During development, parameter fitting was carried out using a compilation of 442 Asp, Glu, His, and Lys residues that had both high-resolution PDB structures and published experimental pKa values available. Applying our PKA17 model, the calculated average unsigned error and RMSD for the residue set were found to be 0.628 and 0.831 pH units, respectively. As a benchmark for comparison, the same residue set was evaluated with the PROPKA software package which resulted in an average unsigned error of 0.761 pH units and an RMSD of 1.063 pH units. Finally, a web interface for the PKA17 software was developed and deployed (http://users.wpi.edu/~jpcvitkovic/pka_calc.html) to make PKA17 available to the wider scientific community.

Every year approximately 2800 Swedes are diagnosed with malignant melanoma, the form of cancer that is most rapidly increasing in incidence in the Western world. The earlier we can identify and diagnose a malignant melanoma, the better is the prognosis. In Sweden, 155 000 benign naevi, harmless skin tumours or moles, are surgically excised each year, many of them because melanoma cannot be dismissed by non-invasive methods. The excisions result in substantial medical costs and cause unrest and suffering of the individual patient. For untrained physicians, it is often difficult to make an accurate diagnosis of melanoma, thus a tool that could help to strengthen the diagnosis of suspected melanomas would be highly valuable. This thesis describes the development and assessment of a non-invasive method for early skin cancer detection. Using near infrared (NIR) and skin impedance spectroscopy, healthy and diseased skin of various subjects was examined to develop a new instrument for detecting malignant melanoma. Due to the complex nature of skin and the numerous variables involved, the spectroscopic data were analysed multivariately using Principal Component Analysis (PCA) and partial leas square discriminant analysis (PLS-DA). The reproducibility of the measurements was determined by calculating Scatter Values (SVs), and the significance of separations between overlapping groups in score plots was determined by calculating intra-model distances. The studies indicate that combining skin impedance and NIR spectroscopy measurements adds value, therefore a new probe-head for simultaneous NIR and skin impedance measurements was introduced. Using both spectroscopic techniques it was possible to separate healthy skin at one body location from healthy skin at another location due to the differences in skin characteristics at various body locations. In addition, statistically significant differences between overlapping groups of both age and gender in score plots were detected. However, the differences in skin characteristics at different body locations had stronger effects on the measurements than both age and gender. Intake of coffee and alcohol prior to measurement did not significantly influence the outcome data. Measurements on dysplastic naevi were significantly separated in a score plot and the influence of diseased skin was stronger than that of body location. This was confirmed in a study where measurements were performed on 12 malignant melanomas, 19 dysplastic naevi and 19 benign naevi. The malignant melanomas were significantly separated from both dysplastic naevi and benign naevi. Overall, the presented findings show that the instrument we have developed provides fast, reproducible measurements, capable of distinguishing malignant melanoma from dysplastic naevi and benign naevi non-invasively with 83% sensitivity and 95% specificity. Thus, the results are highly promising and the instrument appears to have high potential diagnostic utility.

2009/2010
“There is plenty of room at the bottom”. These were the famous words of Richard P. Feynman in 1959 that led to the birth of nanotechnology and nanoscience. Electronic devices based on inorganic semiconductors have been part of our daily lives for the last 60 years. Their miniaturisation has occurred gradually over the years, however, according to Moore’s law the contemporary microelectronic industry’s “top-down” manufacturing technique will soon reach its limits. Therefore, the recent development and increased knowledge of organic semiconductors has led to a tendency to explore alternative avenues with a focus on the creation of electronic devices based on organic molecules. The invention of techniques such STM (1981) and AFM (1986) have facilitated this research, allowing the imaging and manipulation of surfaces and molecules at the nanometre scale (0.1-100 nm). The next step is therefore the development of methods for the controlled fabrication of molecular assemblies and their integration into usable macroscopic systems. In this respect, the “bottom-up” approach offers considerable advantages over any other methodology (i.e. “top-down”) for the construction of nanoscale functional materials and devices. This approach generally exploits the hierarchical self-assembly of functional molecules through multiple non-covalent interactions to prepare long range ordered and defect-free assemblies barely accessible through conventional covalent synthesis. However, an intrinsic drawback of investigating such systems in solution or in a crystal is that molecular components cannot be directly addressed on a nanometric scale. As a consequence, the best engineering methodology involves modifying the surfaces of bulk materials such as metals or semiconductors by deposition of functional organic materials. The modified surfaces are then characterised using scanning probe microscopies (e.g. STM, AFM). To this end, surface-confined, supramolecularly constructed, bi-dimensional (2D) networks, featuring regular porous domains (controllable both in shape and size) are of particular significance in this research domain because their cavities can be used as receptors for the confinement of other remotely controlled functional molecules (e.g. molecular switches, luminescent chromophores). Since these complex nanostructures could ultimately find applications as optoelectronic devices, research efforts in this domain have been gathering momentum in recent years. In Chapter 1, the reader is introduced to the methods employed to construct porous networks on surfaces via supramolecular interactions. The second part of the chapter deals with recent examples of recognition, selection and immobilisation of guest molecules within the cavities of the networks, which is followed in the third part with a discussion about surface assemblies that display structural features or functionality in the third dimension. The last section of the chapter is devoted to the construction of porous networks on surfaces via the interactions of biomimetic molecules (e.g. DNA), which leads to the objectives of the present doctoral project. Inspired by the self-assembly of DNA into nanoporous arrays, it was postulated that the Watson-Crick base pairing of oligonucleotide’s nucleobases would be ideal in preparing 2D porous networks with large receptor cavities. The idea was to covalently attach complementary single stranded oligonucleotides to rigid angular and linear unit core modules respectively, and then allow the two units to self-assemble on surfaces. However, instead of using DNA oligonucleotides, the use of peptide nucleic acid (PNA) oligonucleotides was proposed since more robust architectures would be obtained due to the higher duplex stability displayed by this class of biomimetic molecules. This doctoral dissertation describes the synthetic steps taken towards achieving this goal. The design of the angular and linear units bearing complementary PNA oligomers, required for the preparation of self-assembled nanoporous arrays are described. However, prior to synthesizing these complex molecules, a simpler proof of principle was required to confirm that PNA duplexes could be formed on surfaces and also, whether the presence of chromophoric moieties (e.g. porphyrin) appended to the PNA strands had any effect on duplex formation and duplex stability. The molecule designed for this proof of principle was a self-complementary PNA dodecamer bearing a porphyrin adduct. The synthesis of the self-complementary PNA oligomer required for the preparation of the PNA-porphyrin adduct is described in the first part of Chapter 2. The main synthetic routes and protecting-group strategies used to prepare PNA monomers and oligomers are described first. This is followed by a discussion of the orthogonal protecting group strategies chosen for our project that would allow the isolation of PNA oligomers bearing protected nucleobases following resin-cleavage. This is contrary to the general norm in existing strategies wherein resin-cleavage and nucleobase deprotection is carried out in situ, however, it was required in our synthetic strategy since the terminal amino group of the PNA oligomers was required for further solution phase reactions. To this end, two protecting group strategies were proposed, a Fmoc/Mmt and Fmoc/Cbz-protecting group strategies. The solid support chosen for the Fmoc/Mmt strategy was Tentagel featuring a base-cleavable linker. Due to the failure to hydrolyse the linker during the resin-cleavage step, the Fmoc/Mmt strategy was abandoned. In the second strategy, an acid-cleavable Rink amide resin was chosen as the solid support, therefore a Fmoc/Cbz-protecting group strategy was chosen since it would allow the TFA-mediated cleavage of the oligomer from the resin, without the deprotection of the Cbz groups from the nucleobases. The preparation of the target PNA oligomer (sequence: TTAATTAATTAA) using the Fmoc/Cbz strategy is described in the next section. First, the required monomers for the oligomer synthesis were prepared using established procedures. Then, following reports of the advances in microwave assisted solid phase peptide synthesis claiming improved purity of oligomer products using short coupling times, the solid phase PNA oligomerisation was attempted using microwave irradiation. Three attempts were performed. The first, using a standard laboratory microwave, resulted in a complex mixture of products at the dodecamer stage. An improvement was observed in the results using the CEM discover SPS microwave which was specifically designed for solid phase synthesis, however, the crude dodecamer obtained was still inseparable from the by-products. Similar results were obtained with the CEM liberty microwave, which was an automated solid phase synthesis setup. Finally, utilising manual solid phase synthesis, the target PNA dodecamer was obtained. The HPLC chromatogram of crude PNA dodecamer obtained following resin cleavage displayed a single major product, which was subsequently purified. The oligomer was then deprotected by treatment with TMSI, and was analysed by mass spectrometry, which confirmed that the target dodecamer had been isolated. Section 2.2 described our efforts to prepare PNA-chromophore adducts. Following the isolation of the PNA dodecamer, attempts to covalently attach a porphyrin moiety to the resin-bound oligomer via an amide linkage failed, possibly due to steric hindrance. Subsequently, an azide linker was appended to the oligomer, and attempts to attach an acetylene functionalised porphyrin using a Cu(I)-catalysed 1,3-dipolar cycloaddition were performed. Unfortunately, this approach also did not yield the target adduct. These unsuccessful results paved the way to the development of a Cu(I)-free 1,3-dipolar cycloaddition that enabled the attachment of chromophores to the PNA oligomer. Recently published reports of Cu(I)-free 1,3-dipolar cycloaddition reactions applied on DNA oligomers offered inspiration towards this goal. The reported strategies involved the generation of a nitrile oxide species, which then reacted with either an alkene or an alkyne to form an isoxazoline or an isoxazole. Two methods of generating the nitrile oxide species were evaluated using anthracene derivatives. The first method involved the base-mediated dehydrochlorination of anthracene hydroximoyl chloride to yield the nitrile oxide, which then reacted with a dipolarophile that was introduced into the reaction mixture. The second approach to generating a nitrile oxide species involved treating an O-silylated hydroxamic acid derivative of anthracene with trifluoromethanesulfonic anhydride in the presence of a base (Carreira’s method). Following successful trapping of the nitrile oxides generated by both methods using trimethylsilyl ethylene as the dipolarophile, the reactions were applied on a resin-bound, acetylene-functionalised PNA dodecamer. Both methods yielded the target PNA-anthracene adduct. Since the nitrile oxide-acetylene 1,3-dipolar cycloaddition reaction had never been applied on porphyrins, a method had to be developed. Attempts to prepare a hydroximoyl chloride derivative of a porphyrin resulted in the decomposition of the macrocycle upon treatment with chlorinating agents (NCS, tert-BuOCl, and 1-chlorobenzotriazole), therefore, the hydroximoyl chloride method was abandoned in favour of the Carreira method. An O-silylated hydroxamic acid derivative of porphyrin was synthesized, and upon exposure to trifluoromethanesulfonic anhydride and Et3N, the nitrile oxide was generated and was trapped with a large excess (200 eq.) of trimethylsilyl ethylene yielding the target tetra-isoxazole porphyrin derivative in 62% yield, which corresponded to a yield of 89% per 1,3-dipolar cycloaddition. Optimisation of the reaction conditions using phenyl acetylene as the dipolarophile allowed similar yields to be obtained with only a 10 eq. excess of the acetylene. Having developed a protocol that was compatible with both PNA and porphyrin, the utility of the method to prepare a variety of PNA-chromophore adducts was tested. Hydroxamate derivatives of pyrene, porphyrin, phenanthroline and fluorescein chromophores were prepared. Subsequently, the corresponding nitrile oxide species were generated and were reacted with the resin-bound, acetylene-functionalised PNA dodecamer. The PNA-pyrene adduct was successfully isolated (Figure v), however, the other target PNA-chromophore products were not isolated. The porphyrin nitrile oxide derivative was insoluble in the reaction medium, thus preventing the cycloaddition reaction from proceeding. In the case of the fluorescein hydroxamate, the presence of nucleophilic functional groups in the starting material were probably reactive towards the trifluoromethanesulfonic anhydride reagent, therefore it was unlikely that the nitrile oxide species was formed, and thus the cycloaddition reaction could not proceed. Finally, the reaction with the phenanthroline derivative yielded a new product, however mass spectrometry analysis indicated that it did not correspond the target PNA-phenanthroline adduct. Further work is currently underway to re-evaluate these reactions. In parallel to the synthetic work, a preliminary study into the deposition of PNA onto mica surfaces was investigated using AFM imaging. Deposition of drops of an aqueous solution of deprotected self-complementary PNA dodecamer onto clean mica surfaces using spin coating resulted in aggregates of PNA on the surface. Following annealing of the solution, a repeated deposition of a single drop of the solution resulted in a completely different surface assembly. The surface was saturated by what was thought to be PNA duplexes. This was confirmed by the deposition of drop of a solution that was diluted ten-fold which resulted in an AFM image where bright spot were intermitted by clean mica surface. Topographical analysis of the surface indicated that the bright spots were an average in 1 nm in height, which closely corresponds to the expected height of PNA duplexes, thus confirming that PNA duplexes could be deposited onto surfaces.
XXII Ciclo
1981

Background: The present study considers some of the underlying mechanisms that may be acting in postnatal depression (PND). It has been suggested that rumination predicts problem solving ability and that child temperament and maternal attributions predict mother-infant bonding. This study aims to investigate the role that brooding and reflective rumination may play in predicting and mediating these relationships in postnatal women. Methods: Postnatal women were recruited to complete an online survey.190 women responded and completed the Edinburgh Postnatal Depression Scale (EPDS), Maternal Attribution Scale (MAS), Postpartum Bonding Questionnaire (PBQ), Parental Problem Solving Task (PPST), Rumination Response Scale (RRS), Infant Behaviour Questionnaire (IBQ) and a confidence in problem solving using a Visual Analogue Scale (VAS). Results: Analyses showed that reflective rumination mediated the relationship between low infant soothability and high negative attributions, on maternal problem solving. Reflective and Brooding Rumination also predicted confidence in problem solving and mother-infant bonding. Analyses showed that infant temperament (soothability and distress) and maternal attributions (positive and negative) predicted confidence in problem solving and mother-infant bonding Limitations: This study employed a correlational design and therefore all inferences regarding possible causal pathways are tentative. Limitations include the use of self report measures to assess mother-infant bonding and infant temperament. Additionally the PPST is a new measure which needs further validation. Conclusions: Reflective rumination may act as an adaptive strategy for women in the postnatal period when faced with difficult child temperaments, and for those employing negative attributions, when faced with parent specific problem solving tasks. In addition, Brooding and Reflective Rumination may be important in predicting difficulties in mother-infant bonding. Difficult Infant temperaments and less positive or more negative maternal attributions, may affect problem solving, confidence in problem solving and mother-infant bonding in the postnatal period. Future research should look to replicate these findings and explicate possible causal relationships within a postnatal population.

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CNPQ – Conselho Nacional de Desenvolvimento Científico e Tecnológico
Este estudo utiliza de métodos econométricos para analisar o crime pela ótica da vítima e busca encontrar quais as características pessoais e municipais tornam mais propensas à atração do criminoso no Brasil. A partir de dados disponibilizados pela Pesquisa Nacional de Amostra por Domicílios de 2008 e de 2009, se estima modelos Probit para encontrar os determinantes da vitimização, e da propensão a registrar a ocorrência de três tipos de crime: furto, roubo e agressão física. Os resultados observados apontaram que o perfil do criminoso possui características contrárias ao perfil da vítima, ou seja, o indivíduo criminoso em geral possui baixa escolaridade enquanto o vitimado possui alta escolaridade. Os resultados encontrados mostram que o sexo e o estado civil dos indivíduos são características determinantes para a vitimização por estes crimes. O trabalho conclui que homens solteiros estão mais expostos a violência, o que corrobora com as teorias do estilo de vida e das atividades rotineiras na explicação da vitimização criminal. O trabalho ainda estuda o papel de outros fatores na explicação da violência no Brasil, tais como, rendimentos, idade e escolaridade e as condições macroeconômicas.
This study makes use of econometric methods to analyze crime from the perspective of the victim and seeks to find which personal and local characteristics trigger the attention of criminals in Brazil. From data provided by the National Research of Sample per Household of 2008 and 2009, Probit models are estimated to identify the reasons for victimization, and the likelihood to make a complaint of three types of crime: theft, robbery, and physical assault. Results show that the profile of the perpetrator features characteristics opposed to the profile of the victim, that is, the criminal generally has low education while the victim has high education. The findings also point that gender and marital status of individuals are crucial to victimization characteristics for such crimes. The study concludes that single men are more exposed to violence, which corroborates theories of lifestyle and routine activities in the explanation of criminal victimization. Besides that, the research takes into account the role of other factors in explaining violence in Brazil, such as, income, age and education, as well as macroeconomic conditions.

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Conselho Nacional de Desenvolvimento Científico e Tecnológico
A tese tem como objetivos: (i) avaliar o impacto do Programa Nacional de Alimentação Escolar (PNAE) sobre o nível nutricional; (ii) investigar o efeito da carência e do distúrbio nutricional sobre a defasagem idade-série e sobre o desempenho de escolas públicas brasileiras de 1ª a 8ª séries do ensino fundamental quando avaliados em testes de proficiência. Existe uma carência de estudos no Brasil a respeito dos efeitos da subnutrição sobre o desempenho escolar dos estudantes. Fato que é, em parte, explicado pela carência de dados que avalie de forma representativa o estado nutricional dos estudantes no Brasil. Esta tese, contudo utiliza dados de uma pesquisa recente ASBRAN com informações do perfil nutricional de aproximadamente 20.000 alunos de 1110 escolas públicas brasileiras. A amostra não só representa a população de estudantes de escolas públicas no Brasil, como também as informações levantadas viabilizam o estudo dos objetivos propostos. Completando os dados da ASBRAN são usados dados municipais do IBGE e do IPEA provenientes do Censo 2000 e dados educacionais do INEP 2007. Primeiramente, a tese investiga a relação do aluno com a merenda escolar. Constatou-se, por exemplo, que estudantes com carência nutricional apresentavam maior probabilidade de irem à escola apenas por conta da merenda. Além disso, ficou comprovado que municípios de maior vulnerabilidade apresentam maiores probabilidades de seus alunos irem à escola apenas pela alimentação escolar. A seguir, a tese investiga os efeitos do PNAE sobre o estado nutricional dos alunos das escolas públicas. Para isso, utilizou-se a técnica de Propensity Score. Verificou-se que o PNAE contribui para a melhoria dos desequilíbrios nutricionais registrados pelos alunos de escolas públicas A análise de impacto da carência nutricional (subnutrição) e do distúrbio alimentar sobre a defasagem idade-série envolveu o uso da distribuição binomial negativa pelo fato da variável dependente ser de contagem. Os resultados mostram que a carência nutricional tem impacto direto sobre a defasagem idade-série no Brasil, este efeito é estatisticamente significante a 99 por cento de nível de confiança e permanece inalterado à medida que se adiciona variáveis explicativas que descrevem as condições socioeconômicas das crianças, dos municípios e as características físicas das escolas. Os resultados mostram que os alunos que apresentam distúrbio nutricional apresentam nível menor de defasagem idade-série, contrariando os resultados obtidos por alguns autores. Por outro lado, os resultados das estimações de impacto do PNAE sobre o desempenho da escola não são conclusivos para a maioria das variáveis testadas. No que diz respeito ao desempenho da escola em testes de proficiência, não parece existir correlação entre a performance da instituição de ensino e o estado nutricional do aluno

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Funda????o Jo??o Pinheiro
Inseguran??a alimentar pode ser definida como a limita????o ou a incerteza de se ter acesso a alimentos adequados, em qualidade e quantidade suficientes, sem que essa restri????o possa, necessariamente, afetar as condi????es biol??gicas dos indiv??duos. Neste trabalho, o objetivo central ?? investigar quais os principais fatores associados ?? inseguran??a alimentar e ?? inseguran??a alimentar com fome, tendo como universo de an??lise a Regi??o Nordeste. Os dados utilizados foram provenientes da PNAD 2004, representativos de uma popula????o composta de 12.531.052 domic??lios. Utilizou-se a regress??o log??stica de resposta bin??ria para avaliar os fatores associados ?? inseguran??a alimentar e ?? inseguran??a alimentar com fome. Todas as an??lises foram estratificadas por situa????o censit??ria (rural/urbano). Observou-se, conforme esperado, que a renda domiciliar esteve fortemente associada ?? inseguran??a alimentar e ?? inseguran??a alimentar com fome nos domic??lios da Regi??o Nordeste, independentemente da situa????o censit??ria. Quanto menor a renda, maior a chance de inseguran??a alimentar e de inseguran??a alimentar com fome. Para os domic??lios urbanos a gama de fatores associados ?? inseguran??a alimentar e ?? inseguran??a alimentar com fome s??o em maior n??mero, abrangendo tanto as caracter??sticas do domic??lio, como as da pessoa de refer??ncia. J?? no rural, predominam os fatores diretamente associados ao acesso, preparo e conserva????o dos alimentos (esta ??ltima vari??vel ??? descrita pela presen??a de geladeira ??? apenas para a inseguran??a alimentar). Um resultado que merece ser ressaltado ?? a contribui????o positiva da presen??a de crian??as e adolescentes para que o domic??lio n??o esteja em inseguran??a alimentar com fome, o que pode estar associado a um maior direcionamento dos recursos dispon??veis para garantir as necessidades alimentares dos membros do domic??lio.
Food insecurity is the limitation or uncertainty of having access to adequate food in sufficient quantity and quality. The aim of the present study was to investigate the main factors that were associated with food insecurity and food insecurity with hunger. The analyses were done for the Northeast, Brazil. Data used were from PNAD 2004, representing a population of 12,531,052 households. Logistic regressions of binary response were used in order to evaluate factors associated with food insecurity and food insecurity with hunger. All analyses were stratified by rural/urban situation. Household income was highly and strongly associated with food insecurity and food insecurity with hunger in households in the Northeast, regardless of the rural/urban situation. The lower the income the higher the odds of food insecurity and food insecurity with hunger. For urban households, a higher number of factors (related both to the characteristics of the household and the person in charge of the household) were associated with food insecurity and food insecurity with hunger as compared to urban households. In rural areas, factors directly associated with access, preparation and storage of food revealed significance. A result to be emphasized is that there was a negative association between the presence of children and adolescents and food insecurity with hunger. This finding can be related to the increased targeting of available resources to ensure that the household members could have enough to eat.
Pessoa, fam??lia e sociedade

En l’actualitat hi ha molta controvèrsia en relació a la natura de l’aprenentatge social primat. Una qüestió s’ha focalitzat en les capacitats dels ximpanzés, i si aquesta espècie es capaç d’aprendre de la conducta del modelo (copia de d’accions), o per contra, aprèn indirectament a través de las conseqüències que les acciones d’aquest han general en l’ambient (copia de resultats). L’objectiu principal ha consistit en avaluar de forma experimental l’habilitat en la copia d’accions i/o de resultats en un grup de 14 de ximpanzés allotjats a la Fundació Mona, a través de 11 tasques-problema-que variaven en relació als nivells de dificultat. Els subjectes varen observar diferents tipus d’informació, d’acord a 3 condicions; 1) grup control (sense informació), 2) Només resultats (informació no social), i 3) accions i resultats (informació social) Les demostracions implicaven observar acciones rellevants i irrellevants. Per avaluar els efectes de la informació causal els aparells foren presentats en dos condiciones addicionals, opaca i transparent. Cada participant va rebre 8 intents. En cada intent, la conducta dels subjectes fou avaluada mitjançant; (1) Tipus de manipulacions, (2) Copia de acciones, (3) Èxit 4) Latència (5) Primeres respostes. Per a les tasques complexes, es va analitzar, a mes, la copia d’accions a nivell seqüencies, i el tipus de transicions entre components. Els resultats mostraren efectes globals de l’aprenentatge social en la majoria de variables. Els subjectes foren menys eficients en la condició control que en les experimentals. Globalment no es varen trobar diferencies importants entre les condicions experimentals, on el individus es varen comportar de la mateixa forma independentment del tipus d’informació mostrada, sobre acciones i/o resultats. Sembla que els ximpanzés emulen como estratègia principal. No obstant, l’absència d’imitació no fou total, es varen trobar evidencies de copia d’accions relacionades amb la dificultat de la tasca, però únicament quan les accions foren analitzades en base a la mitjana de tots els intents.
En la actualidad existe mucha controversia sobre la naturaleza del aprendizaje social primate. Una cuestión se ha centrado en las capacidades de los chimpancés, y si esta especie es capaz de aprender directamente de la conducta del modelo (copia de acciones), o por el contrario indirectamente a través de las consecuencias de las acciones de éste (copia de resultados). El objetivo general consistió en evaluar de forma experimental la habilidad de copia de acciones y/o de resultados en un grupo de 14 chimpancés (Pan troglodytes), alojados en la Fundació Mona (Girona), a través de 11 tareas-problema-que variaban en relación a los niveles de dificultad. Los sujetos observaron diferentes tipos de información de acuerdo a 3 condiciones; 1) grupo control (sin información), 2) sólo resultados, sin acciones (información no social), y 3) acciones y resultados (información social). Las demostraciones implicaban la observación de acciones relevantes e irrelevantes. Para evaluar los efectos de la información causal los aparatos fueron presentados en dos condiciones adicionales, una opaca y otra transparente. Cada participante recibió 8 intentos. En cada intento, la conducta de los sujetos fue evaluada mediante; (1) Tipo de manipulaciones, (2) Copia de acciones, (3) Éxito 4) Latencia (5) Primeras respuestas. Para las tareas complejas, se añadieron las variables copia de secuencias y tipo de transiciones entre componentes. Los resultados mostraron efectos globales del aprendizaje social en la mayoría de variables. Los sujetos fueron menos eficientes en la condición control que en las experimentales. Globalmente no se hallaron diferencias importantes entre las condiciones experimentales, donde los individuos se comportaron de la misma forma a expensas de la información mostrada, sobre acciones y/o resultados. De este modo, parece que los chimpancés emulan como estrategia principal. Sin embargo, la ausencia de imitación no fue total, se hallaron evidencias de copia relacionadas con la dificultad de la tarea, pero únicamente cuando las acciones fueron promediadas a través de todos los intentos.
There is currently a controversy about the nature of chimpanzees’ social learning. One of the main issues is focused on chimpanzees’ learning abilities and whether this species is able to learn directly from a model's behaviour (action copy) or otherwise learns indirectly through the consequences of the model’s actions (results copy). The aim of this study was to evaluate the ability to copy actions and /or results on a sample of 14 chimpanzees (Pan troglodytes) from the Fundació Mona through 11 two-target puzzle box- with varying levels of difficulty. The individuals witnessed different sets of demonstrations under three conditions. 1) Control group, without information 2) results only, without actions (non-social information) 3) actions and results (social information). The demonstration involved both causally relevant actions and irrelevant actions, and, in addition, to assess the possible effects of the causal information, the box was presented in each of two conditions, opaque and clear. Each participant received eight trials in which they were given access to the puzzle box for manipulation. Subjects’ behaviour in each trial was assessed for (1) Type of manipulations, (2) Copy actions, (3) Success (4) Latency (5) First responses. For complex tasks the assessed of Copy of sequence order and the number and type of transitions between consecutive actions were added. An overall effect on social learning was detected in many variables. Individuals in the control group were less efficient than individuals in the experimental groups. Overall there were no significant differences between experimental groups, where individuals behave in the same way despite the kind of information they witnessed (social or non-social). Thus, they extract information mainly through the consequences of the model’s actions (emulation) or individually. However, the absence of action copy (imitation) was no total, some coping was found as well related to task difficulty and causal information, but only if the performance was averaged across trials.

Aquesta tesi consta d'un primer capítol introductori, set capítols amb diferents resultats i una bibliografia. El primer capítol conté la definició i els resultats previs necessaris per abordar la resta de la memòria. El capítol 2 i 3 estan molt relacionats. En el primer es descriu un mètode alternatiu per al còmput de les constants de Poincaré--Liapunov. A diferència de mètodes anteriors, el mètode presentat no requereix el càlcul d'integrals i d'ona de forma explícita les constants de Poincaré--Liapunov. En el tercer capítol es descriu com s'ha implementat aquest nou mètode i els resultats que d'ona per a sistemes quadràtics i sistemes amb termes no lineals cúbics homogenis. El quart capítol es centra en equacions d'Abel i la seva integrabilitat. Es descriu la forma d'una integral primera que sigui algebraica en funcíó de les variables dependents i es donen múltiples exemples d'equacions d'Abel integrables en aquest sentit. En el cinquè capítol també s'aborda el problema de la integrabilitat però per a equacions diferencials en el pla definides per funcions analítiques. Es fa un reescalat de les variables dependents i de la variable independent amb un paràmetre "epsilon" que està elevat a potències senceres (blow-up paramètric) de forma que el sistema resultant sigui analític en "epsilon". Es d'ona un mètode que aprofita que una integral primera, si existeix, ha de ser analítica en el paràmetre a fi de trobar condicions per a l'existència d'aquesta integral primera. D'aquesta manera es defineix el que s'anomenen variables essencials del sistema. Els darrers tres capítols versen sobre les equacions d'Abel i el problema del centre. En general es consideren equacions d'Abel trigonomètriques. En el sisè capítol es donen algunes condicions necessàries i suficients per a que una equació d'Abel trigonomètrica definida per polinomis trigonomètrics de grau fins a 3 tingui un centre. Tots els exemples donats en aquest capítol tenen un centre universal. En el capítol setè es d'ona un exemple d'una equació d'Abel trigonomètrica definida per polinomis trigonomètrics de grau 3 que té un centre que no és universal. D'aquesta manera es resol un problema obert: determinar el grau més petit pel qual un equació d'Abel trigonomètrica amb centre no és de composició. El darrer capítol tracta equacions d'Abel trigonomètriques i polinomials i d'ona un compendi dels darrers resultats coneguts i conjectures sobre el problema del centre en aquestes equacions. També es donen exemples nous d'equacions d'Abel amb centre.
Esta tesis consta de un primer capítulo introductorio, siete capítulos con diferentes resultados y una bibliografía. El primer capítulo contiene la definición y los resultados previos necesarios para abordar el resto de la memoria. Los capítulos 2 y 3 están muy relacionados. En el primero se describe un método alternativo para el cómputo de las constantes de Poincaré--Liapunov. A diferencia de métodos anteriores, el método presentado no requiere el cálculo de integrales y da de forma explícita las constantes de Poincaré--Liapunov. En el tercer capítulo se describe cómo se ha implementado este nuevo método y los resultados que da para sistemas cuadráticos y sistemas con términos no lineales cíbicos homogéneos. El cuarto capítulo se centra en ecuaciones de Abel y su integrabilidad. Se describe la forma de una integral primera que sea algebraica en función de las variables dependientes y se dan múltiples ejemplos de ecuaciones de Abel integrables en este sentido. En el quinto capítulo también se aborda el problema de la integrabilidad pero para ecuaciones diferenciales en el plano definidas por funciones analíticas. Se hace un reescalado de las variables dependientes y de la variable independiente con un parámetro "epsilon" que está elevado a poténcias enteras (blow-up paramétrico) de forma que el sistema resultante sea analítico en "epsilon". Se da un método que aprovecha que una integral primera, si existe, debe ser analítica en el parámetro con el fin de encontrar condiciones para la existéncia de esta integral primera. De esta manera se define lo que se llaman variables esenciales del sistema. Los últimos tres capítulos versan sobre las ecuaciones de Abel y el problema del centro. En general se consideran ecuaciones de Abel trigonométricas. En el sexto capítulo se dan algunas condiciones necesarias y suficientes para que una ecuación de Abel definida por polinomios trigonométricos de grado hasta 3 tenga un centro. Todos los ejemplos dados en este capítulo tienen un centro universal. En la capítulo séptimo se da un ejemplo de una ecuación de Abel definida por polinomios trigonométricos de grado 3 que tiene un centro que no es universal. De esta manera se resuelve un problema abierto: determinar el grado mas pequeño por el que una ecuación de Abel trigonométrica con centro no es de composición. El último capítulo trata ecuaciones de Abel trigonométricas y polinomiales y da un compendio de los últimos resultados conocidos y conjeturas sobre el problema del centro en estas ecuaciones. También se dan ejemplos nuevos de ecuaciones de Abel con centro.
This thesis consists of a first introductory chapter, seven chapters with different results and a bibliography. The first chapter contains the definition and the previous results necessary to address the rest of the memory. Chapters 2 and 3 are closely related. In the first one, an alternative method is described for the computation of the Poincaré--Liapunov constants. Unlike previous methods, the presented method does not require the computation of primitives and gives an explicit expression of the Poincaré--Liapunov constants. The third chapter describes how this new method has been implemented and the results that it gives for quadratic systems and systems with homogeneous, cubic, non-linear terms. The fourth chapter focuses on Abel equations and their integrability. We describe the form of a first integral that is algebraic in function of the dependent variables and give more examples of equations of Abel integrable from this point of view. The fifth chapter also discusses the integrability problem but for differential equations in the plane defined by analytical functions. A rescaling of the dependent and the independent variables with a parameter "epsilon" which is elevated to integer powers (parametrical blow up) so that the resulting system is analytical in "epsilon". A method is given that takes advantage that a first integral, if it exists, it must be analytical in the parameter in order to find conditions for the existence of this first integral. In this way we define what are called essential variables of the system. The last three chapters deal with Abel equations and the center problem. In general, we consider Abel trigonometric equations. In the sixth chapter some necessary and sufficient conditions for an Abel equation defined by trigonometric polynomials of degree up to 3 have a center are given. All the examples given in this chapter have a universal center. In the seventh chapter it is given an example of an Abel equation defined by trigonometric polynomials of degree 3 with a center which is not universal. In this way an open problem is solved: to determine the lowest degree such that a trigonometric Abel equation has a center which is not a composition center. The last chapter deals with trigonometric and polynomial Abel equations and gives a survey of the last known results and conjectures about the center problem for these equations. Besides some new examples of Abel differential equations with a center are given.

The fuel consumption of a seagoing vessel is, through a combination of Box-Cox transforms and principal component analysis, reduced to a univariatefunction of the primary principle component with mean model error −3.2%and error standard deviation 10.3%. In the process, a Latin-hypercube-inspired space partitioning sampling technique is developed and successfully used to produce a representative sampleused in determining the regression coefficients. Finally, a formal optimisation problem for minimising the fuel use is described. The problem is derived from a parametrised expression for the fuel consumption, and has only 3, or 2 if simplified, free variables at each timestep. Some information has been redacted in order to comply with NDA restrictions. Most redactions are either names (of vessels or otherwise), units, andin some cases (especially on figures) quantities.

Presentation was performed remotely using Zoom.

Polyhydroxyalkanoates (PHA) are a family of biodegradable polyesters naturally synthesised by some bacteria and archaea. PHA have high industrial value as bioplastics for packaging and biomedical applications. However, their broader use is hindered by high production costs and uncontrolled variation of polymer properties. The extreme halophile Haloferax mediterranei shows bioprocess advantages that can be exploited for the low cost production of the PHA copolymer Poly(3-hydroxbutyrate-co-3-hydroxyvalterate) (PHBV). The focus of this thesis is to identify process variables responsible for the uncontrolled variation of PHA properties in order to progress towards the robust production of PHBV using H. mediterranei. The outcome of the investigation is a novel cultivation strategy for the reliable synthesis of PHBV copolymers with controlled composition and microstructure showing minor differences in material characteristics. Initially, growth kinetics and PHBV synthesis were characterised under nitrogen-excess and nitrogen-limiting conditions in ammonium and for the first time, nitrate. The nitrogen source and concentration influenced PHBV accumulation and variations in polymer composition were observed with ammonium, highlighting the importance of the control of cultivation conditions. Volatile fatty acids (VFA) were found to be a more direct approach to determine PHBV composition and for the first time were used as substrates in H. mediterranei cultures. When the cells were grown in C4:0/C5:0 mixtures, the 3HV fraction in the PHBV was proportional to the percentage of C5:0 in the feed mixture, allowing the synthesis of copolymers with a predefined composition ranging from pure PHB to pure PHV. The cultivation strategy proved effective for the synthesis of HV rich PHBV, which is not easily obtained due to the 3HV precursor toxicity. The polymer microstructure was controlled using different feeding strategies: co-feeding generated random copolymers, while sequential feeding created block and blend copolymers. The synthesis of block copolymers is of interest because the materials show enhanced yield strength and mechanical strength, making such materials more suitable for commodity uses. Bespoke random, block, and blend copolymers with 0−100 mol% 3HV were synthesized and their thermal and mechanical properties studied. Lastly, high temperature cultivation and several surfactants were tested to enhance the production of bespoke PHBV from VFA. PHBV productivity and accumulation was greatly improved in a fed-batch bioreactor fermentation at 37°C with Tween-80 and the maximum PHBV content 58.9% was obtained. The polymers from shake-flasks and from bioreactors showed minor variations in their material properties, demonstrating the scalability and the robustness of the process developed. Further understanding of the different process variables affecting polymer synthesis and composition was gained in this thesis. It is now possible to produce PHBV with controllable composition, microstructure and minor differences in material characteristics. The novel and robust production strategy developed address the bioprocess challenge of minimising the uncontrolled variation of polymer characteristics that is currently hindering the wider use of PHA hence allowing the production of high quality polymers for commodity goods, packaging and biomedical applications.

Esta pesquisa consiste na solução do problema inverso de transferência radiativa para um meio participante (emissor, absorvedor e/ou espalhador) homogêneo unidimensional em uma camada, usando-se a combinação de rede neural artificial (RNA) com técnicas de otimização. A saída da RNA, devidamente treinada, apresenta os valores das propriedades radiativas [ω, τ0, ρ1 e ρ2] que são otimizadas através das seguintes técnicas: Particle Collision Algorithm (PCA), Algoritmos Genéticos (AG), Greedy Randomized Adaptive Search Procedure (GRASP) e Busca Tabu (BT). Os dados usados no treinamento da RNA são sintéticos, gerados através do problema direto sem a introdução de ruído. Os resultados obtidos unicamente pela RNA, apresentam um erro médio percentual menor que 1,64%, seria satisfatório, todavia para o tratamento usando-se as quatro técnicas de otimização citadas anteriormente, os resultados tornaram-se ainda melhores com erros percentuais menores que 0,04%, especialmente quando a otimização é feita por AG.
This research consists in the solution of the inverse problem of radiative transfer for a participating media (emmiting, absorbing and/or scattering) homogeneous one-dimensional in one layer, using the combination of artificial neural network (ANN), with optimization techniques. The output of the ANN, properly trained presents the values of the radiative properties [w, to, p1 e p2] that are optimized through the following techniques: Particle Collision Algorithm (PCA), Genetic Algorithm (GA), Greedy Randomized Adaptive Search Procedure (GRASP) and Tabu Search (TS). The data used in the training are synthetics, generated through the direct problem without the introduction of noise. The results obtained by the (ANN) alone, presents an average percentage error minor than 1,64%, what it would be satisfying, however, for the treatment using the four techniques of optimization aforementioned, the results have become even better with percentage errors minor than 0,03%, especially when the optimization is made by the GA.

Fire debris analysis currently relies on visual pattern recognition of the total ion chromatograms, extracted ion profiles, and target compound chromatograms to identify the presence of an ignitable liquid according to the ASTM International E1618-10 standard method. For large data sets, this methodology can be time consuming and is a subjective method, the accuracy of which is dependent upon the skill and experience of the analyst. This research aimed to develop an automated classification method for large data sets and investigated the use of the total ion spectrum (TIS). The TIS is calculated by taking an average mass spectrum across the entire chromatographic range and has been shown to contain sufficient information content for the identification of ignitable liquids. The TIS of ignitable liquids and substrates, defined as common building materials and household furnishings, were compiled into model data sets. Cross-validation (CV) and fire debris samples, obtained from laboratory-scale and large-scale burns, were used to test the models. An automated classification method was developed using computational software, written in-house, that considers a multi-step classification scheme to detect ignitable liquid residues in fire debris samples and assign these to the classes defined in ASTM E1618-10. Classifications were made using linear discriminant analysis, quadratic discriminant analysis (QDA), and soft independent modeling of class analogy (SIMCA). Overall, the highest correct classification rates were achieved using QDA for the first step of the scheme and SIMCA for the remaining steps. In the first step of the classification scheme, correct classification rates of 95.3% and 89.2% were obtained for the CV test set and fire debris samples, respectively. Correct classifications rates of 100% were achieved for both data sets in the majority of the remaining steps which used SIMCA for classification. In this research, the first statistically valid error rates for fire debris analysis have been developed through cross-validation of large data sets. The error rates reduce the subjectivity associated with the current methods and provide a level of confidence in sample classification that does not currently exist in forensic fire debris analysis.
Ph.D.
Doctorate
Chemistry
Sciences
Chemistry

More and more Swedish municipalities are adopting approaches that target ‘vulnerable EU citizens’ and the ‘passive collection of money’. This thesis analyses begging permits, bans, and other approaches, motivated by the positions supported in Eskilstuna, Katrineholm and Norrköping. The approaches analysed are irrespective of the municipalitiesthat implemented them. This thesis follows a ‘problem’ questioning approach, taking into consideration the context and the system under which the ‘problems’ are constructed and aims to find the implicit and explicit aims of the various approaches.

Cette thèse étudie les processus de création d’activité (PCA) réussis et les pratiques du raisonnement par effectuation (logique effectuelle) combinées ou non avec le raisonnement par causation – logique causale de la théorie des « causes à effet » chez les entrepreneurs. A partir de notre question de recherche : comment crée-t-on une entreprise réussie et comment résout-on les incidents critiques liés à la création d’activité, nous étudions les démarches que les entrepreneurs ont utilisées dans le 10ème arrondissement de Paris pour identifier des données socio-économiques et des caractéristiques territoriales inédites comme ressources et comme contingences pour réussir leurs processus de création d’activité (PCA). Notre problématique de recherche débouche en réalité dans un continuum logique cherchant à comprendre les processus par lesquels ont suivi les projets de création d’activité qui ont réussi ainsi que les méthodes de résolution d’incidents critiques liés à la création d’activité.Par incident, on entend une difficulté (par extension un problème, un obstacle) qui intervient au cours du projet de création et dont les conséquences peuvent être critiques ou graves si elle n’est pas résolue par les créateurs d’activités. Le caractère critique (ou grave) renvoie ici à ce qui n’est pas de leur routine. Cela veut dire que de tel incident peut entraîner l’abandon du processus de création en cours, voire le freiner ou induire à son renoncement. De ce fait, l’on peut supposer que leur survenance peut être liée au contexte du territoire (par exemple, aux facteurs institutionnels ou aux conditions structurelles du lieu d’installation), au type de ressources mobilisées en question ou aux outils et aux méthodes utilisés. Pour répondre à cette question de recherche nous avons retenu trois hypothèses de recherches, à savoir : L’hypothèse H1 qui correspond à la causation (approche décrite comme dominante dans les milieux des créateurs d’entreprise) où elle postule que l’entrepreneur réussit son processus de création d’activité (PCA) s’il est bien formé dans les business schools avec les bons outils (business model) et les bonnes méthodes (analyse de la valeur) qu’il suffit de répliquer ou de dupliquer dans n’importe quel contexte sans tenir compte des situations ou des contingences favorables ou pas. Autrement s’il est suffisamment qualifié et formé dans les milieux académiques. Ensuite vient l’hypothèse H2 de l’effectuation qui fragilise ou caricature l’hypothèse H1 où l’on considère cette fois-ci que l’entrepreneur réussit son (PCA) s’il sait construire une situation d’opportunité inédite ou imprévue du fait des qualités spécifiques qui lui sont propres tel que le charisme, la personnalité et des capacités individuelles rivales (c’est-à-dire des capacités individuelles presque inégales de par leur nature). Ces deux hypothèses H1 et H2 s’inspirent des travaux de Sarasvathy (publication 2001c, a, b et année suivante). Vient enfin l’hypothèse 3 qui intervient dans cette tension entre H1 et H2 pour leur remise en cause. Pour l’hypothèse 3 (Territoire) l’entrepreneur réussit son (PCA) lorsque celui-ci sait intégrer dans son plan de création d’activité le rôle du territoire disposant des ressources facilitantes (et/ou non contraignantes). Autrement dit, un cadre institutionnel habilitant ou des dispositifs facilitants tels que les politiques publiques de développement et de promotion de l’entrepreneuriat sur le territoire ou sur le lieu d’installation des activités en question. Et dans ce cas pour H3, la formation et les qualifications de l’entrepreneur (H1), son charisme et sa personnalité (H2) importent peu
This thesis examines the business creation process (BCP) and successful practices reasoning effectuation (effectual logic) combined or not with the reasoning by causation - causal logic of the theory of "cause and effect" among entrepreneurs. From our research question: how do you create a successful business and how it solves-critical incidents related to business creation, we study the steps that entrepreneurs have used in 10th arrondissement of Paris to identify socioeconomic data and unpublished resources and territorial characteristics such as contingencies to succeed in business creation process (BCP). Our research problem leads actually in a logical continuum seeking to understand the processes that followed the activity creating successful projects and the methods of resolution of critical incidents related to business creation.By incident means a difficulty (by extension a problem, an obstacle) that occurs during the project creation and whose consequences can be critical or severe if it is not resolved by the creators of activities. The critical nature (or severe) here refers to what is not in their routine. This means that such an incident can result in the abandonment of the creative process in progress or slow down or lead to its cessation. Therefore, we can assume that their occurrence may be related to the context of the territory (eg, institutional factors or structural conditions of the installation place), type of resources mobilized in question or to the tools and methods used.To answer this research question we selected three research hypothesis, namely: The H1 hypothesis that matches causation (described as dominant approach in enterprise environments creators) where it assumes that the entrepreneur succeeds its business creation process (BCP) it is well trained in the business schools with the right tools (business model) and the right methods (value analysis) that simply replicate or duplicate in any context regardless of situations or contingencies favorable or not. Otherwise if it is sufficiently trained and qualifed in academic circles. Next comes the H2 hypothesis effectuation that weakens or caricature H1 should we consider this time that the entrepreneur succeeds his (BCP) if he can build a situation of unusual or unexpected opportunity of made specific qualities of its own such as charisma, personality and rival individual capacities (that is to say almost unequal individual capacities in nature). These two hypotheses H1 and H2 are based on the work of Sarasvathy (publication 2001c, a, b and following year). Finally comes the hypothesis 3 which is involved in this tension between H1 and H2 for their questioning. For hypothesis 3 (Territory) the entrepreneur succeeds his (BCP) when it can integrate into its business plan to create the role of territory with facilitative resources (not binding). In other words, an institutional framework enabling or facilitating of devices such as policy development and promotion of entrepreneurship on the territory or in the place of installation of these activities. And in this case for H3, training and entrepreneur's qualifications (H1), charisma and personality (H2) is irrelevant

Chemometrics is a chemical discipline that uses mathematical and statistical methods in order to extract useful information from multivariate chemical data. Moreover, chemometrics is applied to correlate quality parameters or physical properties to analytical instrument data such as calculating pH from a measurement of hydrogen ion activity or a Fourier transform interpolation of a spectrum. Aim of this thesis project is to develop chemometrical strategies for the elaboration and the interpretation of non-selective complex data in order to solve real problems in food, industry and environmental fields.

Extracting foreground objects from an image is a hot research topic. Doing thisfor high quality real world images in real-time on limited hardware such as asmart phone, is a demanding task. This master thesis shows how this problemcan be addressed using Otsu’s method together with Gaussian probability dis-tributions to create classifiers in different colour channels. We also show howclassifiers can be combined resulting in higher accuracy than using only the indi-vidual classifiers. We also propose using inter-class variance together with imagevariance to estimate classifier quality.A data set was produced to evaluate performance. The data set featuresreal-world images captured by a smart phone and objects of varying complex-ity against plain backgrounds that can be found in a typical office or urban space.

Intra- and intercellular information is communicated by posttranslational modifications (PTMs) and protein-protein interactions, transducing information over cell membranes and to the nucleus. A cells capability to respond to stimuli by several highly complex and dynamic signaling networks provides the basis for rapid responses and is fundamental for the cellular collaborations required in a multicellular organism. Having received diverse stimuli, being positioned at various stages of the cell cycle or, for the case of cancer, containing altered genetic background, each cell in a population is slightly different from its neighbor. However, bulk analyses of interactions will only reveal an average, but not the true variation within a population. Thus studies of interacting endogenous biomolecules in situ are essential to acquire a comprehensive view of cellular functions and communication. In situ proximity ligation assay (in situ PLA) was developed to investigate individual endogenous protein-protein interactions in fixed cells and tissues and was later applied for detection for PTMs. Progression of signals in a pathway can branch out in different directions and induce expression of different target genes. Hence simultaneous measurement of protein activity and gene expression provides a tool to determine the balance and progression of these signaling events. To obtain this in situ PLA was combined with padlock probes, providing an assay that can interrogate both PTMs and mRNA expression at a single cell level. Thereby different nodes of the signaling pathway as well as drug effects on different types of molecules could be investigated simultaneously. In addition to regulation of gene expression, protein-DNA interactions present a mechanism to manage accessibility of the genomic DNA in an inheritable manner, providing the basis for lineage commitment, via e.g. histone PTMs. To enable analyses of protein-DNA interactions in situ we developed a method that utilizes the proximity dependence of PLA and the sequence selectivity of padlock probes. This thesis presents new methods providing researchers with a set of tools to address cellular functions and communication in complex microenvironments, to improve disease diagnostics and to contribute to hopefully finding cures.

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The development of this work aims to aims to show the importance of the application of problem situations for the learning of equations and systems of first degree equations with two variables by students of the 8th grade level in paper and pencil environment and observation of two-pan balance. The proposal is to show the application of solving problem which involves first-degree equations with one unknown and systems of first-degree equation with two unknowns, in order to encourage the students to think, state the solution of the problem, try to overcome the learning difficulties, facing challenges which requires a great effort and dedication and find out by themselves the best strategy which must be used in order to solve the problem and, after this, compare and present the algorithms defined for the solution of these concepts. It is also presented the solution of these problems using the twopan balance method, the addition and substitution method, as well as the method of solving equations and equation systems with two unknowns.
O desenvolvimento deste trabalho tem como objetivo mostrar a importância da aplicação de situações-problemas para a aprendizagem de equações e sistemas de equações do primeiro grau com duas variáveis por alunos do 8° ano do ensino fundamental em ambiente papel e lápis e observação de balança de dois pratos. A proposta é mostrar a aplicação de resolução de problemas que envolva equações do primeiro grau com uma incógnita e sistema de equações do primeiro grau com duas incógnitas, a fim de incentivar os alunos a pensarem, encaminharem a solução do problema, tentarem superar as dificuldades de aprendizagem, enfrentarem desafios que exijam grande esforço e dedicação e descubram por si só a melhor estratégia que deve ser utilizada para o problema ser resolvido e, em seguida, comparar e apresentar os algoritmos definidos para a resolução destes conceitos. É apresentada também, a resolução destes problemas usando o método da balança de dois pratos, o método da adição e o método da substituição como método de resolução de equação e sistemas de equações com duas incógnitas.

The work of this thesis was focused on the development of high-performance algorithms for a new generation of molecular descriptors, with many advantages with respect to its predecessors, suitable for diverse applications in the field of drug design, as well as its implementation in commercial grade scientific software (Pentacle). As a first step, we developed a new algorithm (AMANDA) for discretizing molecular interaction fields which allows extracting from them the most interesting regions in an efficient way. This algorithm was incorporated into a new generation of alignmentindependent molecular descriptors, named GRIND-2. The computing speed and efficiency of the new algorithm allow the application of these descriptors in virtual screening. In addition, we developed a new alignment-independent encoding algorithm (CLACC) producing quantitative structure-activity relationship models which have better predictive ability and are easier to interpret than those obtained with other methods.
El trabajo que se presenta en esta tesis se ha centrado en el desarrollo de algoritmos de altas prestaciones para la obtención de una nueva generación de descriptores moleculares, con numerosas ventajas con respecto a sus predecesores, adecuados para diversas aplicaciones en el área del diseño de fármacos, y en su implementación en un programa científico de calidad comercial (Pentacle). Inicialmente se desarrolló un nuevo algoritmo de discretización de campos de interacción molecular (AMANDA) que permite extraer eficientemente las regiones de máximo interés. Este algoritmo fue incorporado en una nueva generación de descriptores moleculares independientes del alineamiento, denominados GRIND-2. La rapidez y eficiencia del nuevo algoritmo permitieron aplicar estos descriptores en cribados virtuales. Por último, se puso a punto un nuevo algoritmo de codificación independiente de alineamiento (CLACC) que permite obtener modelos cuantitativos de relación estructura-actividad con mejor capacidad predictiva y mucho más fáciles de interpretar que los obtenidos con otros métodos.

Tese de doutoramento em Engenharia Biomédica
The gold standard for microbial detection, in clinical settings or in biofilm studies, is the culture method, which is time-consuming, technical demanding and can give inaccurate results. These limitations have driven scientist to the development of new molecular techniques such as fluorescence in situ hybridization (FISH). This technique has been combined with peptide nucleic acid (PNA) molecules, allowing a faster and specific detection of microorganism. In this work, we attempted to evaluate this technology on the detection of relevant bacterial pathogens on food and clinical samples, and on the characterization of multi-species biofilms. As such, the first goal of this thesis was to design, optimize and test for specificity and sensitivity, three new PNA probes to detect Cronobacter spp., Salmonella spp. and Proteus spp. in different samples. Afterwards, the PNA FISH method was compared with the standard methods used to analyze each type of sample. Cronobacter spp. are clinically relevant bacteria because they can cause infections in new-born infants manly due to the consumption of contaminated powdered infant formula (PIF). A PNA probe for the rapid detection of Cronobacter species in PIF was developed and showed experimental specificity and sensitivity both of 100%. Salmonella spp. are well known enteropathogenic bacteria that cause diseases ranging from a mild gastroenteritis to septicaemia, and infection usually occurs due to the consumption of contaminated products. The Salmonella probe was applied to artificially contaminated samples (powdered infant formula and blood) and to natural samples (water and faeces) and probe testing showed specificity and sensitivity values of 100 and 97,6%, respectively. Proteus species are related with the emergence of complicated urinary tract infection (UTI) mainly for catheterized patients (catheter associated UTI’s *CAUTI’s+), which are the most common nosocomial infection. The PNA probe developed for Proteus spp. detection in urine samples showed experimental specificity and sensitivity both of 100%. Regarding the total time required to obtain the results, considering a preenrichment step before the PNA FISH application, this method allowed detection in less than 20 hours for Salmonella spp. and less than 12 hours for Cronobacter spp., even for detection levels of 1 CFU per 10 g or ml and when the target bacteria are outnumbered by other microorganisms. Comparing to the corresponding standard procedures, this represented time savings for both microorganisms of at least 3 days. For Proteus spp. detection, as an enrichment step is not needed, the PNA FISH method was able to detect in approximately 2 hours, as low as 1×104 CFU/mL (a concentration considered indicative of infection for CAUTI’s), which represents time saving of at least 24 hours. The second main goal of this work was to evaluate the PNA FISH performance, using a multiplex approach, on mixed biofilm samples. As such, the PNA FISH method was applied to the characterization of Salmonella enterica/ Listeria monocytogenes/Escherichia coli single, dual and tri-species biofilms in seven different support materials. Results showed that PNA FISH can bring important information not accessed by the other established techniques (culture and crystal violet) especially regarding the spatial distribution (when combined with confocal laser scanning microscopy) and viable but non cultivable stages. Moreover, the PNA FISH data in combination with data from other established techniques, allowed the development of a model for the tri-species biofilm. It was observed that the higher growth rate and exopolysacharide production ability of E. coli led this microorganism to outcompete the other two species resulting on two well defined layers: the top one only with E. coli, and the bottom one with mixed regions of L. monocytogenes and S. enterica. Our results indicate that PNA FISH could be a reliable alternative or complement to the currently used culture-based techniques as it is a very sensitive, specific and rapid method for the detection and location of specific microorganisms. It can be adapted with little effort to different types of samples, even using multiplex approaches, and to microorganisms with different cell wall properties. Finally, it was also demonstrated that PNA FISH is powerful tool for the characterization of multi-species biofilms and that its application can bring important information about inter-species interactions within these structures.
O método padrão para detecção de microrganismos, em ambiente clínico ou no estudo de biofilmes, é o método de cultura, que é demorado, exige técnica e pode levar a resultados ambíguos. Estas limitações levaram os investigadores a desenvolver novas técnicas moleculares como a hibridação fluorescente in situ (FISH). Esta técnica tem sido combinada com moléculas de ácido peptido nucléico (PNA), permitindo uma detecção rápida e específica dos microrganismos alvo. Neste trabalho, procurou-se avaliar a tecnologia de PNA FISH na detecção de bactérias patogénicas em alimentos e amostras clínicas, assim como na caracterização de biofilmes mistos. Como tal, o primeiro objectivo desta tese foi o desenho, optimização e teste de sensibilidade e especificidade de três novas sondas de PNA para detectar Cronobacter spp., Salmonella spp. e Proteus spp., em diferentes amostras. Seguidamente, o método de PNA FISH foi comparado com os métodos padrão usados na análise de cada tipo de amostra. Cronobacter spp. é um género importante a nível clínico porque pode provocar infecções em recém-nascidos. A infecção está geralmente associada ao consumo de fórmulas infantis em pó (PIF) contaminadas. Neste sentido, foi desenvolvida uma sonda de PNA para a detecção rápida de Cronobacter spp. em amostras de PIF, que mostrou valores experimentais de especificidade e sensibilidade de 100%. Salmonella spp. é um género de bactérias enteropatogénicas que causa doenças que vão desde a gastroenterite leve a septicemia. A infecção geralmente ocorre devido ao consumo de produtos contaminados. A sonda de PNA desenvolvida para Salmonella spp. foi aplicada em amostras artificialmente contaminadas (PIF e sangue) e em amostras naturais (água e fezes), e mostrou valores de especificidade e sensibilidade de 100 e 97,6%, respectivamente. As espécies de Proteus estão relacionadas com o aparecimento de infecção do tracto urinário (UTI) complicadas, principalmente em pacientes cateterizados (UTI's associadas a cateteres [CAUTI]), que são as infecções nosocomiais mais comuns. A sonda de PNA desenvolvida para a detecção de Proteus spp. em amostras de urina apresentou especificidade e sensibilidade experimentais, ambas de 100%. Relativamente ao tempo total necessário para obter os resultados da análise por PNA FISH, mesmo considerando um passo de pré-enriquecimento antes da aplicação da técnica, este método permite detectar Salmonella spp. em menos de 20 horas e Cronobacter spp. em menos de 12 horas mesmo para níveis de detecção de 1 UFC por 10 g ou ml de amostra e quando as bactérias alvo estão em concentração muito inferior aos restantes microrganismos. Comparando com os procedimentos padrão correspondentes, esta técnica representa uma poupança de pelo menos três dias para os dois grupos de microrganismos. Para a detecção de Proteus spp., como uma etapa de enriquecimento não é necessária, o método de PNA FISH foi capaz de detectar, em aproximadamente duas horas, 1 × 104 UFC / mL (uma concentração considerada indicativa de infecção para CAUTI’s), o que representa economia de tempo de pelo menos 24 horas. O segundo objectivo deste trabalho foi avaliar o desempenho PNA FISH, utilizando uma abordagem “multiplex” (várias sondas em simultâneo), em amostras de biofilmes mistos. Para tal, o método de PNA FISH foi aplicado na caracterização de amostras de biofilmes simples e mistos, com duas e três espécies, de Salmonella enterica / Listeria monocytogenes / Escherichia coli, em sete materiais diferentes. Os resultados mostraram que a técnica de PNA FISH pode trazer informações importantes que não são acessíveis às outras técnicas já estabelecidas (cultura e violeta de cristal), especialmente no que diz respeito à distribuição espacial (quando combinado com microscopia confocal de varrimento laser) e estados viáveis mas não cultiváveis. Além disso, a combinação dos dados de PNA FISH com dados de violeta de cristal e cultura permitiu o desenvolvimento de um modelo para o biofilme de três espécies. Foi observado que a taxa de crescimento e a elevada capacidade de produção exopolissacarídeos de E. coli permitiu que a concentração deste microrganismo ultrapassasse as restantes espécies resultando em duas camadas bem definidas: a superior só com E. coli, e a camada inferior com regiões mistas de L. monocytogenes e S. Enteritidis. Estes resultados indicam que o método de PNA FISH pode ser uma alternativa viável e/ou complementar às técnicas de cultura actualmente utilizadas, pois é um método muito sensível, específico e rápido na detecção microbiológica. Além disso, pode ser facilmente adaptado a diferentes tipos de amostras, mesmo usando abordagens “multiplex” e para microrganismos com diferentes propriedades de parede celular. Finalmente, foi também demonstrado que o PNA FISH é uma ferramenta poderosa para a caracterização de biofilmes mistos e que a aplicação pode trazer informações importantes sobre as interacções inter-espécies.

Tese de doutoramento em Engenharia Biomédica
Microbial infections are a major cause of hospitalization leading to high morbidity and mortality. An earlier diagnosis can ensure a more rapid treatment, preventing a worse-case scenario. However, ineffective treatments due to inaccurate microorganism identification can lead to hospitalization extension, undesirable side effects, antimicrobial resistance and increased economic costs. Currently, culturing and serologic methods are the methods of choice in clinical diagnosis, but they lack specificity and are time-consuming. PCR based techniques are frequently used, but they are technically-demanding and need DNA manipulation, being prone to contaminations. Therefore, the development of new molecular techniques more specific, reliable and simpler to use is of great concern. Peptide nucleic acid oligonucleotides can be used with fluorescence in situ hybridization as an alternative molecular method to specifically target the rRNA of microorganisms. In the first part of this thesis, it was intended to design and optimize specific probes for the detection of clinical relevant microorganisms, such as, Helicobacter pylori and its clarithromycin resistance and Aspergillus fumigatus detection. With the application of this technique on different cellular morphologies and in different microbial domains it can be definitely proved the capacity of this method to be used as diagnostic method. H. pylori can colonize the human stomach and cause severe gastric diseases. The standard triple therapy where clarithromycin is the main antibiotic used, is given indiscriminately to all symptomatic patients, leading to antimicrobial resistance. In this study three probes were designed, targeting the three most prevalent clarithromycin resistance point mutations (Hp1, Hp2 and Hp3) in the positions A2143G, A2142G and A2142C of the peptidyltransferase region encoded in domain V of the H. pylori 23S rRNA gene. An additional probe targeting susceptible strains (Hpwt) was also developed and tested simultaneously in H. pylori smears with known clarithromycin resistance profile (specificity and sensitivity of 100%). For method validation in gastric biopsies, a retrospective and prospective cohort studies were made where validity indexes such as sensitivity, specificity and likelihood ratios were assessed to test PNAFISH accuracy. In the retrospective assay, full agreement between PNA-FISH and PCR was achieved, but comparing to the reference method based on culture techniques, the specificity was of 90.9% and sensitivity was of 84.2%. In the prospective cohort study comparing to reference method, PNA-FISH demonstrated that can always detect H. pylori resistant and susceptible genotypes (sensitivity and specificity are 100%). Discrepancies detected between positive PNA-FISH results and negative in the reference method can be explained by some culturing method shortcomings, such as difficulty on H. pylori growth in certain media, contaminations and the viable but non-cultivable state of the bacteria (VBNC). Aspergillus fumigatus is a filamentous fungus that produces conidia. When inhaled, conidia can adhere to epithelial lung cells, leading to pulmonary diseases such as Invasive Aspergillosis. The designed probe could discriminate A. fumigatus from other species, presenting practical specificity and sensitivity of 100%. A germination assay was done to assess probe performance for different cells morphology along mold germination. At 0h and 2h the signal was faint due to lack of ribosome content and cell wall structure. From 4h onwards swelling conidia can be observed, and germ tubes start to appear at 6h. Partial and full germination were detected at 8h and 12h, respectively. This germination process was followed by a signal intensity increase that was not uniform throughout the cell, as more intense fluorescence dots near the nucleus appeared. These dots may correspond to increased ribosomal content. Different concentrations of this fungus were applied on artificially contaminated samples for A. fumigatus detection testing. In blood it was possible to detect this mold as early as 6h at concentrations ranging 1x103 - 1x104 cells ml-1. In artificial sputum media it was only possible to detect A. fumigatus from 16h to 24h at concentrations of 1x102 cells ml-1. In addition, and as a final aim of this work, it was used a commercially available PNA-FISH kit to study Escherichia coli and Pseudomonas aeruginosa biofilm interactions simulating urinary catheters conditions, to demonstrate the potential uses of PNA-FISH. These two bacteria are some of the main responsible for catheters associated urinary tract infections, mainly due to catheter biofilm formation. DAPI staining and CFUs counts in silicon coupons embedded in artificial urine, were used in addition to E. coli/P. aeruginosa PNA-FISH® probes to have an overlook of the behavior in terms of biofilm formation capacity of these two bacteria in single and mixed biofilms. In single-species biofilms it was possible to observe that E. coli appears to form more easily biofilm than P. aeruginosa. From all the results for mixed-biofilms it was possible to infer that some sort of interaction between these two species is happening although P. aeruginosa seems to benefit the most (Log 7 CFU of P. aeruginosa/cm2, comparing to Log 6 CFU/cm2 obtained in pure cultures) comparing to E. coli that decreased cell concentration in presence of the other counterpart. The PNA-FISH combined with CLSM could indeed confirm P. aeruginosa outcompeting E. coli in dual-species biofilms. This probably happens due to P. aeruginosa secreting substances that can be toxic to cultivable E. coli cells, preventing them to proliferate. In fact, although this is the main causative agent of urinary infections for its constant presence in infection places and for its capacity of forming singlespecies biofilm, in the presence of other bacteria seems to lose that capacity. The whole results from this thesis indicate that PNA-FISH is a reliable technique for the specific identification of microorganisms or its genotypic characteristics and it can be used as a method of diagnosis ready to use, in clinical practice or as a mean for studying important microorganism’s mechanisms in research laboratories. It can also be used to discriminate populations in biofilms, as the uncharged backbone of the PNA molecule allows it to diffuse more freely through the biofilm matrix providing a more comprehensive view of the biofilm structure. This feature may allow the more effective development of new eradication therapies.
As infecções causadas por microrganismos são uma das maiores causas de hospitalização, que conduzem a graus de morbilidade e mortalidade elevados. Um diagnóstico precoce assegura que o tratamento é mais efectivo, prevenindo o pior cenário. No entanto, os tratamentos são por vezes ineficientes devido à dificuldade de identificar o microrganismo causador de doença, o que levam a maiores períodos de internamento, efeitos secundários indesejáveis, aumento de resistências a substâncias antimicrobianas e aos aumentos dos custos associados. Actualmente, os métodos mais utilizados em diagnóstico clínico são os métodos de cultura e serológicos, mas apresentam algumas limitações pois são muito demorados e pouco sensíveis. Os métodos baseados em PCR também são frequentemente utilizados mas são tecnicamente exaustivos e como envolvem extracção de ADN, estão sujeitos, muitas vezes, a contaminações. Assim, tem sido dada importância ao desenvolvimento de novas técnicas moleculares mais específicas, seguras e mais fáceis de usar. Sondas de ácido péptido nucleico (PNA) têm sido usadas em associação com hibridação fluorescente in situ (FISH) como um método molecular alternativo por se ligarem de um modo específico ao rARN de microrganismos. A primeira parte do trabalho destinou-se ao desenho e optimização de sondas específicas para detecção de microrganismos clinicamente relevantes como a Helicobacter pylori e a sua resistência à claritromicina e detecção do Aspergillus fumigatus. Com a aplicação desta técnica em diferentes morfologias celulares e nos diferentes domínios microbianos fica definitivamente provada a capacidade deste método para ser usado como método de diagnóstico. A H. pylori é uma bactéria colonizadora do estômago humano, que pode causar doenças gástricas severas. A terapia utilizada mais comum é a terapia tripla onde a claritromicina é o antibiótico mais comummente usado, e é administrada indiscriminadamente a todos os pacientes que apresentam sintomas levando a um elevado grau de resistência deste antibiótico. Neste estudo, foram desenhadas três sondas relativas às três mutações pontuais que conferem resistência à claritromicina (Hp1, Hp2 e Hp3) que se encontram nas posições A2143G, A2142G e A2142C da região peptidiltransferase do domínio V do 23S rRNA. Adicionalmente foi desenhada outra sonda que se liga a estirpes susceptíveis a este antibiótico (Hpwt). Estas sondas foram testadas em esfregaços contendo estirpes de H. pylori com perfil de resistência à claritromicina conhecido (especificidade e sensibilidade de 100%). Para validação da precisão do método em biopsias gástricas foram feitos estudos cohort retrospectivo e prospectivo, onde se testaram alguns índices de validade como a sensibilidade, a especificidade e razões de verosimilhança. No teste retrospectivo, verificou-se concordância total entre o PNA-FISH e o PCR, mas comparando-se os resultados com o teste de cultura de referência a especificidade foi de 90.9% e a sensibilidade de 84.2%. No teste prospectivo e comparando com o método de referência, o PNA-FISH demonstrou que consegue sempre detectar H. pylori com resistência ou susceptibilidade à claritromicina (sensibilidade e especificidade são 100%). As discrepâncias entre os resultados que deram positivo para o PNA-FISH, mas não para o método de referência, podem ser explicadas por algumas lacunas apresentadas pelos métodos de cultura, como por exemplo, o facto de ser por vezes difícil de fazer crescer a H. pylori em certos meios, contaminações e os diferentes estados de cultivabilidade da bactéria (VBNC). O Aspergillus fumigatus é um fungo filamentoso que produz conídios. Quando inalados, os conídios aderem às células epiteliais do pulmão, originando doenças pulmonares como a Aspergilose Invasiva. A sonda desenhada foi capaz de descriminar as estirpes de A. fumigatus de entre todas as estudadas, apresentando especificidade e sensibilidade práticas de 100%. Foram feitos ensaios de germinação de modo a verificar se o desempenho da sonda seria idêntico nas diferentes estruturas do fungo, que aparecem ao longo do processo de germinação. Nas 0h e nas 2h o sinal apresentou-se fraco devido ao baixo conteúdo ribossomal e à estrutura da parede celular dos conídios. A partir das 4h, pôde-se observar a dilatação dos conídios e às 6h o início do aparecimento de tubos germinativos. Às 8h e às 12h foi possível observar-se germinação parcial e total, respectivamente. Todo o processo germinativo foi seguido de um aumento de sinal de fluorescência, que no entanto, não era uniforme em toda a célula, mas com pontos mais fortes perto do núcleo, que poderão corresponder a um aumento do conteúdo ribossomal. Foram aplicadas concentrações seriadas de A. fumigatus em amostras artificialmente contaminadas para detecção do fungo. No sangue foi possível de detectar este fungo logo às 6h, a concentrações compreendidas entre 1x103 -1x104 cells ml-1. Em meio de expectoração artificial foi apenas possível detectá-lo entre as 16h e as 24h a concentrações de 1x102 cells ml-1. No último objectivo deste trabalho, um kit multiplex de sondas de PNAFISH foi utilizado para estudar as interacções entre Escherichia coli e Pseudomonas aeruginosa em biofilmes, simulando as condições em cateteres urinários, como forma de demonstrar os potenciais usos desta técnica. Estas duas bactérias são duas das mais frequentemente responsáveis por infecções do tracto urinário associadas a cateteres, especialmente devido à formação de biofilmes nestes dispositivos médicos. O método de coloração por DAPI e a contagem de unidades formadoras de colónias, em cupões de silicone imersos em urina artificial foram usados em conjunto com as sondas do kit E. coli/P. aeruginosa PNA-FISH® para se obter uma visão generalizada do comportamento destas duas bactérias em biofilmes simples e mistos. Em biofilmes simples é possível observar que a E. coli parece formar biofilme mais facilmente que a P. aeruginosa. Relativamente aos biofilmes mistos, os resultados parecem indicar que existe uma interacção entre estas duas bactérias. No entanto, enquanto a P. aeruginosa parece beneficiar da presença da E. coli (Log 7 CFU de P. aeruginosa/cm2, comparando com Log 6 CFU/cm2 obtidos em culturas puras), a E. coli decresce a sua concentração celular na presença da outra espécie constituinte. A técnica de PNA-FISH em combinação com microscopia confocal (CLSM) pode, de facto, confirmar a superação da P. aeruginosa sobre a E. coli em biofilmes de duas espécies. Este fenómeno provavelmente ocorre pela secreção, pela P. aeruginosa, de substâncias que podem ser tóxicas para a E. coli, impedindo a sua proliferação. Assim, embora a E. coli seja o agente infeccioso mais comum em infecções urinárias pela sua presença constante nos locais de infecção e pela capacidade de formar biofilme simples, na presença de outra bactéria, parece perder essa capacidade. No total, os resultados desta tese indicam que o PNA-FISH é uma técnica segura, adequada para a identificação específica de microrganismos ou suas características genotípicas e que pode ser usada como um método de diagnóstico disponível em prática clínica ou como meio de estudar importantes mecanismos dos microrganismos em laboratórios de pesquisa. Esta técnica também poderá ser usada para descriminar populações em biofilmes devido à estrutura não carregada da molécula de PNA que lhe permite uma melhor difusão na matriz do biofilme, o que levará a uma visão mais esclarecedora acerca da estrutura dos biofilmes. Esta particularidade poderá permitir o estudo de novas terapias de erradicação.
A tese de doutoramento aqui apresentada foi suportada por uma bolsa de doutoramento financiada pela Fundação para a Ciência e Tecnologia (FCT) com a referência SFRH/BD/38124/2007 financiada pelo Programa Operacional Potencial Humano- Quadro de Referência Estratégico Nacional (POPH – QREN) - Tipologia 4.1 - Formação Avançada, comparticipado pelo Fundo Social Europeu (FSE) e por fundos nacionais do Ministério da Ciência, Tecnologia e Ensino Superior (MCTES).

The aim of the studies presented in this thesis was to explore various materials and platforms for the detection of DNA in order to develop a cost-effective, repeatable and reproducible electrical/electrochemical DNA sensor. Chapter 1 is a review chapter consisting of a comparative study of the fundamental working principle, fabrication process, characteristics and performance of different types of DNA biosensors and a detailed discussion on the progress in the development of DNA biosensors. The studies described in Chapter 2 focuses on the development of a flexible, label-Free DNA Sensor using Platinum oxide (PtO) as the sensing element. PtO (100nm) deposited using an optimized reactive ion sputtering process revealed p-type semiconducting behaviour with a band-gap of 1.5 eV, resistivity of 0.16 Ω-m and activation energy of 0.22 eV. XPS spectra indicated the presence of PtO phase (32%) along with PtO2 phase (68%). The XRD spectra indicated the formation of α-PtO2 phase. Arrays of simple, two terminal sensors were fabricated on transparent, flexible, acetate substrates with platinum oxide thin film forming the active layer (8.0 mm X 60 μm) for DNA detection. The sensor operated on the principle of conductance change resulting from the change in charge carrier density due to attachment of DNA to the platinum oxide surface. The DNA attachment onto platinum oxide was experimentally verified by performing Fourier Transform Infrared Spectroscopy (FTIR) and optical fluorescence measurements. The binding constant of DNA to platinum oxide was found to be 7.35 p.m. for every percentage increase in fluorescence intensity. The sensor arrays showed a DNA concentration-dependent current change that was linear over a large dynamic range and sensitivity down to 0.5 nM. The label-free platinum oxide DNA sensors showed reproducibility with a coefficient of variation (CoV) of less than 10%. In chapter 2 reactively sputtered Platinum oxide thin film was used as a DNA sensing element. In chapter 3, we subject the reactively sputtered Platinum oxide thin films to an additional RIE step for 3, 6 and 9 minutes and carry out a detailed comparative study of the material and electrical properties of these films. XRD and XPS analysis revealed that when the reactively sputtered Platinum oxide film was subjected to RIE step for longer periods of time, it became progressively α-PtO2 in nature. Activation energies of 0.24 eV, 0.26 eV, 0.29 eV and 0.31 eV were obtained for the as deposited film and the films subjected to RIE step for 3, 6 and 9 minutes respectively. The Hall mobility of the as deposited Platinum oxide film was found to be 32.15 cm2V-1s-1 at room temperature. However, when the as deposited film was subjected to RIE step for 9 minutes the mobility value rises to as high as 136.13 cm2V-1s-1 at room temperature. In chapter 4, the development of an electrochemical, sequence specific DNA hybridization sensor using platinum microelectrodes is discussed. A new transduction hybridization pathway obtained by coupling electrochemical adsorption and long-range electron transfer through double-stranded DNA is presented. This property was used to investigate long-range electron transfer properties of synthetic oligonucleotides and plasmids layers. A check complementarity assay of two non-labelled short DNA, without heating or DNA labelling, in a 10 minutes protocol, was in fine established. In chapter 5, the possibility of DNA sensing using impedance spectroscopy of layer-by-layer self-assembly of weak polyelectrolytes is explored. Interdigitated capacitors (IDCs) with active area of 1X1 mm2 and electrode spacing of 5 μm are fabricated on acetate sheets for this purpose. Measurement results indicate that there is differential binding of DNA to differently charged polyelectrolyte terminating layer on the IDC surface. The differential binding of DNA to Poly (Allylamine Hydrochloride) (PAH) and Poly (Acrylic acid) was also confirmed using Fluorescence microscopy and FTIR spectroscopy.

Electrochemical impedance measurements were used for the detection of single-strand DNA sequences using a peptide nucleic acid (PNA) probe layer immobilized onto Si/SiO 2 chips. In our approach, the PNA is covalently linked to the surface of Si/SiO 2 chips that have been functionalized with a silane, 3-glycidoxypropyltrimethoxysilane (GPTS). The functionalization procedure has been optimized to ensure maximum available sites for probe attachment. The PNA probe is hybridized with complementary solution-phase target DNA. Impedance measurements allow for the detection of the changes in charge distribution at the oxide/solution interface following modifications to the oxide surface. Due to these modifications, there are significant shifts in the semiconductor's flat-band potential after immobilization and hybridization. The results obtained using this direct and rapid approach are supported by fluorescence measurements according to classical methods for the detection of nucleic acid sequences. One of the main challenges to achieving highly reproducible and more sensitive silicon-based sensor devices is the optimization of the probe layer immobilization procedures. Hybridization may be kinetically or sterically hindered at high surface probe densities. Varying the amount of time that the solid support is exposed to the PNA solution and controlling the probe solution concentration can control the probe density of the probe layer. Fifteen minutes for immobilization appears to be sufficient enough to obtain a single strand layer with a good balance between density and steric hindrance. The second focus of this thesis is the determination of the melting temperature (T m ) of a complex sequence PNA 10-mer by measuring the impedance of the electrochemical system at different temperatures. The T m for perfect matched duplexes is 52.2 ± 0.3{493}C. This compared well to the theoretical one of 50{493}C. The introduction of a single base mismatch in the complementary DNA oligomer results in a 13{493}C lowering of the observed T m . The T m for a single base mismatch is 39.7 ± 0.6{493}C.

Niniejsza praca poświęcona została specyfice przekładu epopei narodowej jako gatunku literackiego. Punkt wyjścia dla podjętych tu rozważań stanowi popełnione przez autorkę rozprawy trzecie w serii przekładów tłumaczenie pierwszej części argentyńskiej epopei narodowej pod tytułem „Martín Fierro” na język polski. Dziewiętnastowieczny poemat pióra Joségo Hernándeza, wpisujący się w nurt tak zwanej literatury gauczowskiej, zmusza do zastanowienia się nad szerokim kontekstem społeczno-politycznym ówczesnej Argentyny, którego Agata J. Kornacka nie przemilcza, świadoma jego znaczenia w świetle kolejnych założeń dysertacji. Dużo miejsca i uwagi poświęca kwestii epopeiczności poematu, zestawieniu komparatystycznemu tłumaczonego dzieła z Mickiewiczowskim „Panem Tadeuszem”, jak również samemu procesowi translacji, z uwypukleniem najważniejszych problemów napotkanych na poziomie strukturalnym i leksykalno-semantycznym. Zasadniczą część pracy, będącą niejako odpowiedzą na postawiony w tytule rozprawy problem badawczy, stanowi próba zdefiniowania przekładu epopei narodowej na tle szerszego kontekstu przekładu poetyckiego, szczegółowy opis strategii i technik translatorskich zastosowanych w tłumaczeniu „Martína Fierro” oraz szereg dodatkowych obserwacji, które w przekonaniu autorki rozprawy pozwalają odróżnić „dobry” przekład epopei narodowej od przekładu „nieudolnego”, choć oba te pojęcia pozostają – i pozostaną – aksjomatycznie rozmyte. Agata J. Kornacka nie umniejsza przy tym znaczenia najważniejszych postulatów współczesnej myśli translatorskiej, choć traktuje je z pewną rezerwą. Niejako na drugim planie rozprawy przedstawiono spostrzeżenia o charakterze ogólnym tudzież uogólniającym oraz wnioski dotyczące kondycji tłumacza literatury, a w szczególności tłumacza poezji, jego mesjanistycznej roli pośrednika w dialogu międzykulturowym i kontrowersyjnego zagadnienia nieprzekładalności.
This doctoral thesis is devoted to the specificity of translating national epics as a literary genre. The starting point for the considerations discussed herein is the translation of the first part of the Argentinian national epic entitled "Martín Fierro" into Polish, committed by Agata J. Kornacka as the third one in a series of transltions. The nineteenth-century poem by José Hernández, part of the so-called gaucho (“gauchoesque”) literature, forces us to reflect on the wide socio-political context of Argentina in those times, a question that is not silenced by the author of this dissertation, aware of its importance in the light of her subsequent assumptions. Much space and attention is devoted to the epic character of the poem, the comparative juxtaposition of the translated work with Mickiewicz's "Pan Tadeusz", as well as the translation process itself, taking into consideration the most important problems encountered at the structural and lexical-semantic level. The main part of the work, which in a way answers the research problem put forward in the title, is an attempt to define the translation of national epics against a wider context of poetry translation, a detailed description of the translation strategies and techniques used in the translation of “Martín Fierro” and a set of additional observations that the author believes make it possible to distinguish between a "good" translation of a national epic from an "inefficient" one, although both of these concepts remain – and will remain – axiomatically blurred. Agata J. Kornacka does not diminish the significance of the most important postulates of contemporary translation studies, although she treats them with a slight skepticism. In the background of this dissertation readers can find general observations and conclusions about the condition of a literary translator, in particular as it comes to translating poetry, his messianic role as an intermediary in intercultural dialogue and the controversial issue of non-translatability.

碩士
國立雲林科技大學
工業工程與管理系
103
Abstract The Taguchi method is one of the methods commonly used in the industry. The workflows of today’s business community belong to complex manufacturing processes. The business community not only explores single quality characteristics but also often requires multiple quality characteristics. Therefore, errors may result from using only the Taguchi method to find the optimal combination of parameters between the various variables of multiple quality characteristics. Factors such as different variables or multiple units can cause problems. Therefore, this study investigated the situation of multiple variables and multiple quality characteristics of products. First, the principal component analysis method was used to merge associated variables in the production of multiple variables without losing the original variables and the independent relationship between each main component. After the variables were simplified, VIKOR (VlseKriterijumska Optimizacija I Kompromisno Resenje, Serbian for Multicriteria Optimization and Compromise Solution) was used to find the best evaluation value between each variable. Finally, each evaluation value was then nested into the Taguchi method of multiple product quality characteristics to find the best combination of parameters. In this study, first find out the quality characteristics of PQL, and then use principal component analysis to identify the main ingredient, and finally to find a comprehensive index from VIKOR, to obtain the best combination of parameters for A_2 B_2 C_3 D_1 E_2 F_3. Key words: Taguchi methods、Principal Component Analysis、VIKOR

The present work describes new aspects of organic and supramolecular chemistry. The scientific contribution consists of two parts, which focus on the development of receptors for the sulfate anion and quantitative assessment of stacking interactions between an anthracene dye and nucleobases in an aqueous solution. In Chapter 1, basic concepts concerning supramolecular chemistry and recognition of cations and anions are discussed, as well as modern methods for the determination of binding constants. Particular attention is paid to fluorescence sensing of ions and underlying mechanisms of binding-induced fluorescence responses. Chapter 2 is dedicated to the design and synthesis of new fluorescent sulfate receptors functioning in aqueous solution. After a short review of the most effective sulfate receptors/probes created so far, a new design of PET probes for sulfate sensing is presented. The syntheses and anion binding properties of new compounds are described. The experimental data obtained for the receptors are discussed in detail to reveal the origin of high selectivity towards sulfate. Chapter 3 explores the importance of nucleobase–arene stacking interactions in recognition of nucleotides by synthetic receptors. Various experimental and theoretical approaches are presented to assess dispersion interactions between aromatic rings and nucleobases in the receptor–nucleotide complexes.

We propose a new method to perform functional principal component analysis (FPCA) for discretely observed functional data by solving successive optimization problems. The new framework can be applied to both regularly and irregularly observed data, and to both dense and sparse data. Our method does not require estimates of the individual sample functions or the covariance functions. Hence, it can be used to analyze functional data with multidimensional arguments (e.g. random surfaces). Furthermore, it can be applied to many processes and models with complicated or nonsmooth covariance functions. In our method, smoothness of eigenfunctions is controlled by directly imposing roughness penalties on eigenfunctions, which makes it more efficient and flexible to tune the smoothness. Efficient algorithms for solving the successive optimization problems are proposed. We provide the existence and characterization of the solutions to the successive optimization problems. The consistency of our method is also proved. Through simulations, we demonstrate that our method performs well in the cases with smooth samples curves, with discontinuous sample curves and nonsmooth covariance and with sample functions having two dimensional arguments (random surfaces), repectively. We apply our method to classification problems of retinal pigment epithelial cells in eyes of mice and to longitudinal CD4 counts data. In the second part of this dissertation, we propose a sparse Fisher’s discriminant analysis method with thresholded linear constraints. Various regularized linear discriminant analysis (LDA) methods have been proposed to address the problems of the LDA in high-dimensional settings. Asymptotic optimality has been established for some of these methods when there are only two classes. A difficulty in the asymptotic study for the multiclass classification is that for the two-class classification, the classification boundary is a hyperplane and an explicit formula for the classification error exists, however, in the case of multiclass, the boundary is usually complicated and no explicit formula for the error generally exists. Another difficulty in proving the asymptotic consistency and optimality for sparse Fisher’s discriminant analysis is that the covariance matrix is involved in the constraints of the optimization problems for high order components. It is not easy to estimate a general high-dimensional covariance matrix. Thus, we propose a sparse Fisher’s discriminant analysis method which avoids the estimation of the covariance matrix, provide asymptotic consistency results and the corresponding convergence rates for all components. To prove the asymptotic optimality, we provide an asymptotic upper bound for a general linear classification rule in the case of muticlass which is applied to our method to obtain the asymptotic optimality and the corresponding convergence rate. In the special case of two classes, our method achieves the same as or better convergence rates compared to the existing method. The proposed method is applied to multivariate functional data with wavelet transformations.