Le présent travail de thèse repose sur la conception d'un système miniaturisé de type biopuce capable d'assurer la capture et le relargage contrôlé de différentes populations des cellules sanguines (e.g. lymphocytes). Ce projet a pour objectif la construction d'un outil potentiel de recherche dans le domaine de l'immunologie ainsi que du diagnostic qui permettrait non seulement de réaliser des essais à partir d'une faible quantité d'échantillon, mais aussi de réduire le temps d'analyse. L'approche consiste plus précisément en la fabrication d'une matrice d'oligonucléotides et l'immobilisation de cellules via une molécule hybride composée d'un anticorps IgG couplé à une séquence d'oligonucléotide complémentaire. La synthèse du produit conjugué est mise en place et conduit à l'assemblage functionnel sur biopuce. Une fois les cellules spécifiquement capturées sur la surface, deux voies de rélargage contrôlé sont explorées. Ainsi, les lymphocytes sont libérées de façon contrôlée et séquentielle par clivage enzymatique d'ADN ou alors par désorption physique possible grâce au chauffage localisé. La détection se fait en temps réel par l'imagerie de la résonance plasmonique de surface (Surface Plasmon Resonance Imaging, SPRi) qui présente l'avantage de pouvoir suivre les phénomènes biomoléculaires en absence de marquage et d'apporter une réponse simultanée d'un échantillon biologique sur un grand nombre des sondes. Accessoirement, une approche instrumentale particulière nous permet d'observer les étapes de capture/relargage par microscopie optique classique. La construction de la biopuce permet également l'élargissement à plusieurs cibles et ouvre ainsi la voie à de nombreuses possibilités d'exploration en termes d'application pour l'analyse d'échantillons biologiques plus complexes tels que du sang. / This PhD thesis is devoted to conception of a miniaturized system of biochip type able to realize a controlled capture and release of different populations of the blood cells (e.g. lymphocytes). The main objective of the project is to create a potential tool of research, especially in the field of immunology, and medical diagnostics as well, that could perform short-time analyses by using a small sample amount. The approach relies more precisely on fabrication of a DNA matrix and further immobilization of cells through a hybrid molecule composed of an IgG antibody covalently coupled with short oligonucleotide sequence. Synthesis of the conjugated product is developed and demonstrates functional assembly on the micro-platform. Lymphocytes are specifically addressed onto biochip surface and once they are captured, two independent strategies of selective release are proposed. Therefore, immobilized cells are specifically detached either upon enzymatic cleavage of oligonucleotide substrate or physically desorbed by local heating and denaturation of double stranded DNA. The system makes use of Surface Plasmon Resonance Imaging (SPRi) to enable real time detection of different biomolecular phenomena in a label-free and high-throughput manner. Accessorily, a particular instrumental approach is developed in order to observe cell capture-release steps directly under optical microscopy. The biochip construction permits to extend its performance to many targets and may be further explored in terms of application to analysis of complex biological samples such as blood.

The aim of this paper is to study whether there is a correlation between how subject pronouns are introduced in Spanish teaching and the pupils' difficulties regarding both their use and the understanding of verb conjugation. What is being investigated is: a) to what extent and in what contexts subject pronouns appear in Spanish textbooks, and b) how secondary education teachers of Spanish work with subject pronouns in the classroom. My conclusion is that the subject pronouns are under-represented in Spanish textbooks, probably due to the interpretation of the null subject parameter and Spanish as a pro-drop language. As far as the work with subject pronouns in the classroom is concerned, a more in-depth analysis needs to be carried out in order to be able to demonstrate a reliable correlation between the teaching method and the students' results. / Syftet med detta arbete är att studera om det finns ett samband mellan hur subjektspronomina introduceras i spanskaundervisningen och elevernas svårigheter kring både användandet av dessa och förståelsen för verbböjning. Det som undersöks är: a) i vilken utsträckning och i vilka sammanhang förekommer subjektspronomina i läromedel för undervisning i spanska, och b) hur högstadielärare i spanska arbetar med subjektspronomina i klassrummet. Min slutsats är att subjektspronomina finns underrepresenterade i läroböcker i spanska, förmodligen på grund av den tolkning som görs av nollsubjektsparametern och spanska som pro-drop språk. Vad gäller arbetet med subjektspronomina i klassrummet behöver en mer fördjupad analys genomföras för att det ska kunna påvisas ett tillförlitligt samband mellan undervisningsmetod och elevernas resultat.

Cette thèse consiste en une étude théorique et expérimentale de la dynamique d'une diode laser à émission latérale (850 nm) avec rétroaction optique à conjugaison de phase. Le dispositif expérimental est conçu afin de voir la diversité dynamique du laser à travers l'analyse temporelle et spectrale alors que le taux de rétroaction varie. La rétroaction à conjugaison de phase est effectuée par mélange à quatre ondes dans un cristal photoréfractif. Le temps de propagation du faisceau laser dans la cavité externe est appelé retard. Sous l’effet de la rétroaction, le système montre une grande richesse dynamique qui varie du chaos total à des régimes auto-pulsés aux fréquences déterminés par le longueur de la cavité externe pour la première fois mis en évidence ici. Des simulations menées en parallèle basées sur les équations de Lang-Kobayashi couramment utilisées apportent des confirmations théoriques aux observations expérimentales. Les principaux points traités sont la crise chaotique et bistabilité de solutions pulsées, les régimes auto-pulsés (stabilisation et déstabilisation) ainsi que les transitions entre eux, la caractérisation d’événements extrêmes de différentes natures avec la distribution statistique associée ainsi qu’une résonance cohérente déterministe de fluctuations à basse fréquence induite par le retard. Au-delà de l’intérêt fondamental de ces résultats et des comparaisons qui peuvent être établies avec d’autres systèmes laser, des applications dans la génération de signaux tout-optique ainsi que le contrôle du chaos sont des débouchés potentiels de cette étude. / This thesis is a theoretical and experimental study of the dynamics of an edge-emitting laser diode (850 nm) with phase-conjugate feedback. The experimental device is designed to see the dynamical range of the laser through the temporal and spectral properties while the feedback rate varies. Phase-conjugate feedback is performed through four-wave mixing in a photorefractive crystal. The propagation time of the laser beam in the external cavity is termed external time delay. Under the effect of the feedback, the system shows a wide dynamical range including chaos and self-pulsing states which characteristic properties are determined by the length of the external cavity. For the first time self-pulsing states at frequencies multiples of the fundamental frequency of the external cavity are evidenced. Simulations carried out based on the commonly-used Lang-Kobayashi laser rate equations provide theoretical confirmations to the experimental observations. The main topics tackled here are chaos crisis and bistability of pulsing solutions, self-pulsing regimes (through their stabilization and destabilization) and the transitions between them, characterization of extreme events of two kinds along with their statistical distribution and delay-induced deterministic coherence resonance of low frequency fluctuations. Beyond the fundamental interest of these results and the many comparisons that can be made with other laser systems, applications in the field of all-optical signal generation and control of chaos are direct consequences of this study.

A partir de uma base teórica que cruza textos advindos do pensamento complexo (Filosofia), do paradigma funcional (Lingüística) e de autores diversos das áreas de Didática, Sociologia e Lingüística Aplicada, afinados de alguma maneira com o paradigma ou pensamento complexo, analisaremos a forma de como a conjugação verbal dos verbos \"ser\" e \"ter\" é introduzida em nosso corpus - quatro livros didáticos de português (do Brasil) para estrangeiros de grande circulação no sul e sudeste do País, e tentaremos obter por meio dessa análise uma visão do quanto cada obra de nosso corpus se aproxima de nossa base teórica. Tal base, resumida em \"quesitos\" no capítulo 5 a serem satisfeitos ou não pelo corpus analisado nos capítulos 6 e 7, encara o indivíduo/aprendiz como um ser extremamente complexo pois portador de nuances as mais diversas, que se não consideradas no processo de aquisição da linguagem, aquisição por si só também extremamente complexa, corremos o risco de se ver esse indivíduo frustrado em sua tentativa legítima de aprender o português como língua estrangeira. A título de sugestão de melhorias na forma como nosso corpus introduz e sedimenta os verbos \"ser\" e \"ter\", descreveremos como os equivalentes desses verbos no inglês são introduzidos pela série New Interchange, além, e principalmente, de oferecer sugestões de melhorias inspiradas em nossa base teórica e em nossa experiência na área de ensino de Português Língua Estrangeira. / From a theory basis that crosses texts from the complex paradigm (Philosophy), the functional paradigm (Linguistics) and several other authors from Didactics, Sociology and Applied Linguistics, anyhow affined with the paradigms above, we will make an analysis of the way that the verb conjugation of \"ser\" (to be) and \"ter\" (to have) is presented by our corpus - four Portuguese as foreign language books largely sold in the South and Southeast of Brazil - and try to visualize through this analysis in which degree each of these books gets close to our theory basis. The result of this \"texts crossing\" is summarized in quests form in chapter 5 to be met or not by our corpus analyzed in chapters 6 and 7. Such theory basis considers the individual/learner as an extremely complex being, a carrier of many different aspects that, once not taken into account in the language acquisition process, such acquisition extremely complex itself, one takes the risk to have this individual frustrated in his legitimate try to learn Portuguese as a foreign language. As an improvement suggestion on how verbal conjugation of \"ser\" and \"ter\" could be presented by the Portuguese as foreign language books focused in this dissertation, we will describe how the verb conjugation of \"be\" and \"have\" is presented by the New Interchange series, besides offering improvement suggestions inspired by our theory basis and by our own experience in the field of teaching Portuguese as foreign language.

In this thesis will be described research towards the development of bioorthogonal bond-cleavage reactions, and their applications in targeted drug delivery (Figure 1). The first project relates to the development of a palladium mediated bond-cleavage or "decaging" reaction which can cause a propargyl carbamate to decompose and release an amine. This was further developed by the incorporation of a protein modification handle which allowed an amine-bearing drug to be covalently ligated to a protein by a palladium-cleavable linker. This chemistry was demonstrated by the conjugation of the anticancer drug doxorubicin to a tumour targeted anti-HER2 nanobody. The drug could then be delivered to cancer cells upon addition of a palladium complex. The second project relates to the development of a platinum mediated bond-cleavage reaction. This was developed with the aim of using platinum-containing anticancer drugs - such as cisplatin - as a catalyst to cause drug release reactions in tumours. In this reaction an alkyne-containing amide can decompose to release an amine upon addition of platinum complexes, and was applied to the release of prodrugs of the cytotoxins monomethylauristatin E and 5-fluorouracil in cancer cells. A cisplatin-cleavable antibody-drug conjugate was designed and synthesised, and progress towards its biological evaluation will be discussed.

La majorité des ADC qui sont actuellement en clinique et en développement sont produits par une conjugaison chimique via les résidus lysine ou cystéine, menant à un produit hétérogène pour leur ratio toxine sur anticorps (DAR). L'objet des travaux de thèse a pour but de décrire la caractérisation in vitro et in vivo de nouveaux ADC optimisés et construits à partir de l'anticorps anti-CD30 cAC10, ayant le même squelette polypetidique que Adcetris, et de comparer les résultats à ce dernier. La transglutaminase bactérienne (BTG) a été utilisée pour conjuguer de manière site-spécifique la MMAE aux glutamines aux positions 295 et 297 du cAC10, amenant à des ADCs homogènes de DAR 4, TG-ADC. Des travaux préliminaires ont permis d’établir les conditions optimales de conjugaison avec un procédé en deux étapes. Les tests de cytotoxicité ont révélé des EC50 comparables entre Adcetris et les TG-ADC. Les données d’efficacité in vivo montrent une efficacité équivalente voire légèrement supérieure pour les TG-ADC que Adcetris. L'étude de biodistribution in vivo dans un modèle avec et sans tumeur est réalisé avec un 125-I TG-ADC et est comparé à 125I-Adcetris. Le TG ADC site spécifique montre une meilleure distribution tumorale. Adcetris a une distribution non médiée par la cible, dans le foie et la rate, plus importante. En ligne avec ces résultats, la dose maximale tolérée des TG ADC est significativement plus élevée que Adcetris chez le rat. Ces résultats suggèrent que les ADC homogènes ont une meilleure pharmacocinétique et un meilleur index thérapeutique comparés aux ADC avec des DAR hétérogènes. / Most ADC that are currently in clinical use or development produced by chemical conjugation of a toxin via either lysine or cysteine residues, inevitably leading to heterogeneous products with variable drug-to-antibody ratios (DARs). Here, we describe the in vitro and in vivo characterization of novel ADCs that are based on the anti-CD30 antibody cAC10, which has the same polypeptide backbone as Adcetris, and compare the results with the latter. Bacterial transglutaminase (BTG) was exploited to site-specifically conjugate derivatives of MMAE to the glutamines at position 295 and 297 of cAC10, yielding homogeneous ADCs with a DAR of 4, TG-ADC. Preliminary works have led to define optimal conditions for conjugation, but also define a two step process. In vitro cell toxicity experiments revealed comparable EC50-values for Adcetris and TG-ADC. The efficacy data have shown slightly better efficacy for TG-ADC compared to Adcetris. Quantitative time-dependent in vivo biodistribution studies in normal and xenografted mice were performed with a selected 125I TG ADC and compared with 125I-Adcetris. Adcetris has an higher liver and spleen unspecific uptakes. In line with these results, the maximum tolerated dose of the BTG-coupled ADC (> 60 mg/kg) was significantly higher than that of ADCETRIS® (18 mg/kg) in rats. These results suggest that homogenous ADCs display improved pharmacokinetics and better therapeutic indexes compared to chemically modified ADCs with variable DARs.

Negli ultimi decenni l'utilizzo degli antibiotici a scopo terapeutico o come promotori della crescita nell'allevamento animale ha portato alla comparsa e alla diffusione di microrganismi resistenti. In questo contesto, la presenza di Lattobacilli (LAB) antibiotico resistenti non rappresentano di per sé un rischio clinico. Tuttavia la possibilità che essi ma possono essere veicolo di geni codificanti l'antibiotico-resistenza verso batteri patogeni presenti negli alimenti o nel tratto gastro-intestinale umano (inclusi enterococchi, streptococchi e listeria), costituisce un possibile rischio per la salute umana che deve essere attentamente valutato. Obiettivo di questo lavoro è stato quello di valutare attraverso metodi di indagine fenotipica con le tecniche delle microdiluizioni in brodo, Etest e disc-diffusion, i livelli di antibiotico resistenza per le specie S. thermophilus e L. plantarum verso gli antibiotici tetraciclina, eritromicina, clindamicina, streptomicina, gentamicina, ampicillina. Ceppi atipici appartenenti alla specie S. thermophilus sono stati sottoposti ad analisi genetiche con lo scopo di caratterizzare e localizzare i geni responsabili della resistenza. E' stato inoltre testato il possibile trasferimento orizzontale dei geni di antibiotico resistenza nativi da S. thermophilus verso i batteri Gram-positivi E. faecalis e Listeria monocytogenes. In alcuni ceppi di S. thermophilus resistenti si sono infine osservati e studiati particolari caratteri fenotipici ( fitness ) correlati alla presenza delle determinanti genetiche di antibiotico resistenza nell'ospite batterico. / In the last decades, the use of antibiotics in human therapy or in animal husbandry as growth promoters has induced the development and the diffusion in antibiotic resistant micro-organisms. In this context antibiotic resistant Lactic Acid Bacteria (LAB) do not represent a clinical risk in themselves. However, the possibility that S. thermophilus cultures might transfer antibiotic resistance genes to pathogenic species either present in food or in the gastrointestinal tract (including enterococci, streptococci and listeria) represents a potential clinical risk that needs to be carefully evaluated. The aim of this study was to evaluate by means of phenotypic methods (microdilution, E-test, disc-diffusion) the levels of antibiotic resistance for S. thermophilus and L. plantarum species against the antibiotic tetracycline, erythromycin, clyndamicin, streptomycin, gentamycin and ampicillin. The atypical resistant S. thermophilus strains were subjected to genetic analyses in order to characterise and to localise the antibiotic resistance determinants. Furthermore the ability of the resistant S. thermophilus strains in transferring the antibiotic resistant determinant was assessed in mating experiments using as recipients the Gram-positive bacteria E. faecalis and Listeria monocytogenes. In same resistant S. thermophilus strains, special bacterial fitness related with the presence of the antibiotic resistance determinants in the bacterial hosts were observed and studied.

One the most daunting technical challenges in the realization of biosensors is functionalizing transducing surfaces for the detection of biomolecules. Functionalization is defined as the formation of a bio-compatible interface on the transducing surfaces of bio-chemical sensors for immobilizing and subsequent sensing of biomolecules. The kinetics of functionalization reactions is a particularly important issue, since conventional functionalization protocols are associated with lengthy process times, from hours to days. The objective of this thesis is the improvement of the functionalization protocols and their kinetics for biosensing applications. This objective is realized via modeling and experimental verification of novel functionalization techniques in microfluidic environments. The improved functionalization protocols using microfluidic environments enable in-situ functionalization, which reduces the processing times and the amount of reagents consumed, compared to conventional methods. The functionalization is performed using self-assembled monolayers (SAMs) of thiols. The thiols are organic compounds with a sulphur group that assists in the chemisorption of the thiol to the surface of metals like gold. The two reactions in the functionalization process examined in this thesis are the SAM formation and the SAM/probe molecule conjugation. SAM/probe molecule conjugation is the chemical treatment of the SAM followed by the binding of the probe molecule to the SAM. In general, the probe molecule is selective in binding with a given biomolecule, called the target molecule. Within this thesis, the probe molecule is an antibody and the target molecule is an antigen. The kinetics of the reaction between the probe (antibody) and the target biomolecule (antigen) is also studied. The reaction between an antigen and its antibody is called the immunoreaction. The biosensing technique that utilizes the immunoreaction is immunoassay. A numerical model is constructed using the finite element method (FEM), and is used to study the kinetics of the functionalization reactions. The aim of the kinetic studies is to achieve both minimal process times and reagents consumption. The impact of several important parameters on the kinetics of the reactions is investigated, and the trends observed are explained using kinetic descriptive dimensionless numbers, such as the Damköhler number and the Peclet number. Careful numerical modeling of the reactions contributes to a number of findings. A considerably faster than conventional SAM formation protocol is predicted. This fast-SAM protocol is capable of reducing the process times from the conventional 24-hours to 15 minutes. The numerical simulations also predict that conventional conjugation protocols result in the overexposure of the SAM and the probe molecule to the conjugation reagents. This overexposure consequently lowers conjugation efficiencies. The immunoreaction kinetics of a 70 kilo-Dalton heat shock protein (HSP70) with its antibody in a hypothetical microchannel is also investigated through the FEM simulations. Optimal reaction conditions are determined, including the flow velocity and the surface concentration of the immobilized probes (antibodies). Based on the numerical results and a series of experimental studies, the fast-SAM protocol application is successfully confirmed. Moreover, the optimum reagent concentration for a given one- hour conjugation process time is determined. This functionalization protocol is successfully applied to immobilize the HSP70 antibody on gold surfaces. The use of the fast-SAM protocol and the predicted optimum conjugation conditions result in binding of the HSP70 antibody on gold, with the same or superior immobilization quality, compared to the conventional protocols. Upon implementation of a 70 μm.s^(-1) flow velocity, the reaction is observed to complete in around 30-35 minutes, which is close to the numerically predicted 30 minutes and 16 seconds. This immunoreaction time is considerably less than conventional 4-12 hour processes. The modified in-situ functionalization techniques achieved here are promising for substantially reducing the preparation times and improving the performance of biosensors, in general, and immunoassays, in particular.

Unconventional nanofabrication techniques; both those which have been newly developed and those under development, had brought inexpensive, facile, yet high quality means to fabricate nanostructures that have feature sizes of less than 100 nm in industry and academia. This dissertation focuses on developing unconventional fabrication techniques, building studying platforms, and studying the mechanisms behind them. The studies are divided into two main facets and four chapters. The first facet, in Chapter II and Chapter III, deals with the research and development of different nanofabrication techniques and nanostructures. These techniques include litho-synthesis, colloidal lithography, and photolithography. The nanostructures that were fabricated by these techniques include the metal nanoparticle arrays, and the self-assembled CdSe nanoring arrays. At the same time, the dissertation provides mechanisms and models to describe the physical and chemical nature of these techniques. The second area of this study, in Chapter III to Chapter V, presents the applications of these nanostructures in fundamental studies, i.e. the mechanisms of plasmon enhanced fluorescence and photo-oxidation kinetics of CdSe quantum dots, and applications such as molecular sensing and material fabrication. More specifically, these applications include tuning the optical properties of CdSe quantum dots, biomodification of CdSe quantum dots, and copper ion detection using plasmon and photo enhanced CdSe quantum dots. We have successfully accomplished our research goals in this dissertation. Firstly, we were able to tune the emission wavelength of quantum dots, blue-shifted for up to 45 nm, and their surface functionalization with photo-oxidation. A kinetic model to calculate the photo-oxidation rates was established. Secondly, we established a simple mathematical model to explain the mechanism of plasmon enhanced fluoresce of quantum dots. Our calculation and experimental data support the fluorescence resonance energy transfer (FRET) mechanism between quantum dots and the metal nanoparticles. Thirdly, we successfully pattered the CdSe quantum dots (diameter ~4 nm) into nanorings with tunable diameters and annular sizes on different substrates. We also established a physical model to quantitatively explain the mechanism with the forces that involved in the formation of the nanorings.

No description available.