The stomach is frequently exposed to hazardous agents and to resist this harsh environment, several protective mechanisms exist. Of special interest is the gastric pathogen Helicobacter pylori which causes gastritis, ulcers and cancer but the mechanism leading to these diseases are still unclear. However it is very likely that H. pylori negatively influence the protection mechanisms that exist in the stomach. The aims of the present investigation were first to develop an in vivo mouse model in which different protection mechanisms could be studied, and second to investigate the influence of H. pylori on these mechanisms. An in vivo preparation of the gastric mucosa in mice was developed. This preparation allows studies of different gastric mucosal variables and can also be applied for studies in other gastro-intestinal organs. Mice chronically infected with H. pylori, were shown to have a reduced ability of the mucosa to maintain a neutral pH at the epithelial cell surface. This could be due to the thinner inner, firmly adherent mucus gel layer, and/or to defective bicarbonate transport across the epithelium. The Cl-/HCO3- exchanger SLC26A9 was inhibited by NH4+, which also is produced by H. pylori. The mRNA levels of SLC26A9 were upregulated in infected mice, suggesting a way to overcome the inhibition of the transporter. Furthermore, the hyperemic response to acid pH 2 and 1.5 was abolished in these mice. The mechanisms by which the bacteria could alter the blood flow response might involve inhibition of the epithelial iNOS. Water extracts of H. pylori (HPE) reduces the blood flow acutely through an iNOS and nerve-mediated pathway, possibly through the endogenous iNOS inhibitor ADMA. Furthermore, HPE alters the blood flow response to acid as the hyperemic response to acid pH 0.8 is accentuated in mice treated with HPE.

In this work, the problem of detecting neuronal spikes or action potentials (AP) in noisy recordings from a Microelectrode Array (MEA) is investigated. In particular, the spike detection algorithms should be less complex and with low computational complexity so as to be amenable for real time applications. The use of the MEA is that it allows collection of extracellular signals from either a single unit or multiple (45) units within a small area. The noisy MEA recordings then undergo basic filtering, digitization and are presented to a computer for further processing. The challenge lies in using this data for detection of spikes from neuronal firings and extracting spatiotemporal patterns from the spike train which may allow control of a robotic limb or other neuroprosthetic device directly from the brain. The aim is to understand the spiking action of the neurons, and use this knowledge to devise efficient algorithms for Brain Machine Interfaces (BMIs). An effective BMI will require a realtime, computationally efficient implementation which can be carried out on a DSP board or FPGA system. The aim is to devise algorithms which can detect spikes and underlying spatio-temporal correlations having computational and time complexities to make a real time implementation feasible on a specialized DSP chip or an FPGA device. The time-frequency localization, multiresolution representation and analysis properties of wavelets make them suitable for analysing sharp transients and spikes in signals and distinguish them from noise resembling a transient or the spike. Three algorithms for the detection of spikes in low SNR MEA neuronal recordings are proposed: 1. A wavelet denoising method based on the Discrete Wavelet Transform (DWT) to suppress the noise power in the MEA signal or improve the SNR followed by standard thresholding techniques to detect the spikes from the denoised signal. 2. Directly thresholding the coefficients of the Stationary (Undecimated) Wavelet Transform (SWT) to detect the spikes. 3. Thresholding the output of a Teager Energy Operator (TEO) applied to the signal on the discrete wavelet decomposed signal resulting in a multiresolution TEO framework. The performance of the proposed three wavelet based algorithms in terms of the accuracy of spike detection, percentage of false positives and the computational complexity for different types of wavelet families in the presence of colored AR(5) (autoregressive model with order 5) and additive white Gaussian noise (AWGN) is evaluated. The performance is further evaluated for the wavelet family chosen under different levels of SNR in the presence of the colored AR(5) and AWGN noise. Chapter 1 gives an introduction to the concept behind Brain Machine Interfaces (BMIs), an overview of their history, the current state-of-the-art and the trends for the future. It also describes the working of the Microelectrode Arrays (MEAs). The generation of a spike in a neuron, the proposed mechanism behind it and its modeling as an electrical circuit based on the Hodgkin-Huxley model is described. An overview of some of the algorithms that have been suggested for spike detection purposes whether in MEA recordings or Electroencephalographic (EEG) signals is given. Chapter 2 describes in brief the underlying ideas that lead us to the Wavelet Transform paradigm. An introduction to the Fourier Transform, the Short Time Fourier Transform (STFT) and the Time-Frequency Uncertainty Principle is provided. This is followed by a brief description of the Continuous Wavelet Transform and the Multiresolution Analysis (MRA) property of wavelets. The Discrete Wavelet Transform (DWT) and its filter bank implementation are described next. It is proposed to apply the wavelet denoising algorithm pioneered by Donoho, to first denoise the MEA recordings followed by standard thresholding technique for spike detection. Chapter 3 deals with the use of the Stationary or Undecimated Wavelet Transform (SWT) for spike detection. It brings out the differences between the DWT and the SWT. A brief discussion of the analysis of non-stationary time series using the SWT is presented. An algorithm for spike detection based on directly thresholding the SWT coefficients without any need for reconstructing the denoised signal followed by thresholding technique as in the first method is presented. In chapter 4 a spike detection method based on multiresolution Teager Energy Operator is discussed. The Teager Energy Operator (TEO) picks up localized spikes in signal energy and thus is directly used for spike detection in many applications including R wave detection in ECG and various (alpha, beta) rhythms in EEG. Some basic properties of the TEO are discussed followed by the need for a multiresolution approach to TEO and the methods existing in literature. The wavelet decomposition and the subsampled signal involved at each level naturally lends it to a multiresolution TEO framework at the same time significantly reducing the computational complexity due the subsampled signal at each level. A wavelet-TEO algorithm for spike detection with similar accuracies as the previous two algorithms is proposed. The method proposed here differs significantly from that in literature since wavelets are used instead of time domain processing. Chapter 5 describes the method of evaluation of the three algorithms proposed in the previous chapters. The spike templates are obtained from MEA recordings, resampled and normalized for use in spike trains simulated as Poisson processes. The noise is modeled as colored autoregressive (AR) of order 5, i.e AR(5), as well as Additive White Gaussian Noise (AWGN). The noise in most human and animal MEA recordings conforms to the autoregressive model with orders of around 5. The AWGN Noise model is used in most spike detection methods in the literature. The performance of the proposed three wavelet based algorithms is measured in terms of the accuracy of spike detection, percentage of false positives and the computational complexity for different types of wavelet families. The optimal wavelet for this purpose is then chosen from the wavelet family which gives the best results. Also, optimal levels of decomposition and threshold factors are chosen while maintaining a balance between accuracy and false positives. The algorithms are then tested for performance under different levels of SNR with the noise modeled as AR(5) or AWGN. The proposed wavelet based algorithms exhibit a detection accuracy of approximately 90% at a low SNR of 2.35 dB with the false positives below 5%. This constitutes a significant improvement over the results in existing literature which claim an accuracy of 80% with false positives of nearly 10%. As the SNR increases, the detection accuracy increases to close to 100% and the false alarm rate falls to 0. Chapter 6 summarizes the work. A comparison is made between the three proposed algorithms in terms of detection accuracy and false positives. Directions in which future work may be carried out are suggested.

The entorhinal cortex (EC) in the medial temporal lobe plays a critical role in memory formation and is implicated in several neurological diseases including temporal lobe epilepsy and Alzheimer’s disease. Despite the known importance of this brain region, little is known about the normal bioelectrical activity patterns of the EC in awake, behaving primates. In order to develop effective therapies for diseases affecting the EC, we must first understand its normal properties. To contribute to our understanding of the EC, I monitored the activity of individual neurons and populations of neurons in the EC of rhesus macaque monkeys during free-viewing of photographs using electrophysiological techniques. The results of these experiments help to explain how primates can form memories of, and navigate through, the visual world. These experiments revealed neurons in the EC that represent visual space with triangular grid receptive fields and other neurons that prefer to fire near image borders. These properties are similar to those previously described in the rodent EC, but here the neuronal responses relate to viewing of remote space as opposed to representing the physical location of the animal. The representation of visual space may be aided by another EC neuron type that was discovered, free-viewing saccade direction cells, neurons that signaled the direction of upcoming saccades. Such a signal could be used by other cells to prepare to fire according to the future gaze location. Many of these spatially-responsive neurons also represented memory for images, suggesting that they may be useful for associating items with their locations. I also examined the neuronal circuitry of recognition memory for visual stimuli in the EC, and I found that population synchronization within the gamma-band (30-140 Hz) in superficial layers of the EC was modulated by stimulus novelty, while the strength of memory formation modulated gamma-band synchronization in the deep layers and in layer III. Furthermore, the strength of connectivity in the gamma-band between different layers was correlated with the strength of memory formation, with deep to superficial power transfer being correlated with stronger memory formation and superficial to deep transfer correlated with weaker memory formation. These findings support several previous investigations of hippocampal-entorhinal connectivity in the rodent and advance our understanding of the functional circuitry of the medial temporal lobe memory system. Finally, I explored the design of a device that could be used to investigate properties of brain tissue in vitro, potentially aiding in the development of treatments for disorders of the EC and other brain structures. We designed, fabricated, and validated a novel device for long-term maintenance of thick brain slices and 3-dimensional dissociated cell cultures on a perforated multi-electrode array. To date, most electrical recordings of thick tissue preparations have been performed by manually inserting electrode arrays. This work demonstrates a simple and effective solution to this problem by building a culture perfusion chamber around a planar perforated multi-electrode array. By making use of interstitial perfusion, the device maintained the thickness of tissue constructs and improved cellular survival as demonstrated by increased firing rates of perfused slices and 3-D cultures, compared to unperfused controls. To the best of our knowledge, this is the first thick tissue culture device to combine forced interstitial perfusion for long-term tissue maintenance and an integrated multi-electrode array for electrical recording and stimulation.

Optogenetics is a set of technologies that enable optically triggered gain or loss of function in genetically specified populations of cells. Optogenetic methods have revolutionized experimental neuroscience by allowing precise excitation or inhibition of firing in specified neuronal populations embedded within complex, heterogeneous tissue. Although optogenetic tools have greatly improved our ability manipulate neural activity, they do not offer control of neural firing in the face of ongoing changes in network activity, plasticity, or sensory input. In this thesis, I develop a feedback control technology that automatically adjusts optical stimulation in real-time to precisely control network activity levels. I describe hardware and software tools, modes of optogenetic stimulation, and control algorithms required to achieve robust neural control over timescales ranging from seconds to days. I then demonstrate the scientific utility of these technologies in several experimental contexts. First, I investigate the role of connectivity in shaping the network encoding process using continuously-varying optical stimulation. I show that synaptic connectivity linearizes the neuronal response, verifying previous theoretical predictions. Next, I use long-term optogenetic feedback control to show that reductions in excitatory neurotransmission directly trigger homeostatic increases in synaptic strength. This result opposes a large body of literature on the subject and has significant implications for memory formation and maintenance. The technology presented in this thesis greatly enhances the precision with which optical stimulation can control neural activity, and allows causally related variables within neural circuits to be studied independently.

Les biofilms sont des communautés de microorganismes incorporés dans une matrice exo-polymérique complexe. Ils sont reconnus pour jouer un rôle important comme barrière de diffusion dans les systèmes environnementaux et la santé humaine, donnant lieu à une résistance accrue aux antibiotiques et aux désinfectants. Comme le transfert de masse dans un biofilm est principalement dû à la diffusion moléculaire, il est primordial de comprendre les principaux paramètres influençant les flux de diffusion. Dans ce travail, nous avons étudié un biofilm de Pseudomonas fluorescens et deux hydrogels modèles (agarose et alginate) pour lesquels l’autodiffusion (mouvement Brownien) et les coefficients de diffusion mutuels ont été quantifiés. La spectroscopie par corrélation de fluorescence a été utilisée pour mesurer les coefficients d'autodiffusion dans une volume confocal de ca. 1 m3 dans les gels ou les biofilms, tandis que les mesures de diffusion mutuelle ont été faites par cellule de diffusion. En outre, la voltamétrie sur microélectrode a été utilisée pour évaluer le potentiel de Donnan des gels afin de déterminer son impact sur la diffusion. Pour l'hydrogel d'agarose, les observations combinées d'une diminution du coefficient d’autodiffusion et de l’augmentation de la diffusion mutuelle pour une force ionique décroissante ont été attribuées au potentiel de Donnan du gel. Des mesures de l'effet Donnan (différence de -30 mV entre des forces ioniques de 10-4 et 10-1 M) et l'accumulation correspondante d’ions dans l'hydrogel (augmentation d’un facteur de 13 par rapport à la solution) ont indiqué que les interactions électrostatiques peuvent fortement influencer le flux de diffusion de cations, même dans un hydrogel faiblement chargé tel que l'agarose. Curieusement, pour un gel plus chargé comme l'alginate de calcium, la variation de la force ionique et du pH n'a donné lieu qu'à de légères variations de la diffusion de sondes chargées dans l'hydrogel. Ces résultats suggèrent qu’en influençant la diffusion du soluté, l'effet direct des cations sur la structure du gel (compression et/ou gonflement induits) était beaucoup plus efficace que l'effet Donnan. De même, pour un biofilm bactérien, les coefficients d'autodiffusion étaient pratiquement constants sur toute une gamme de force ionique (10-4-10-1 M), aussi bien pour des petits solutés chargés négativement ou positivement (le rapport du coefficient d’autodiffusion dans biofilm sur celui dans la solution, Db/Dw ≈ 85 %) que pour des nanoparticules (Db/Dw≈ 50 %), suggérant que l'effet d'obstruction des biofilms l’emporte sur l'effet de charge. Les résultats de cette étude ont montré que parmi les divers facteurs majeurs qui affectent la diffusion dans un biofilm environnemental oligotrophe (exclusion stérique, interactions électrostatiques et hydrophobes), les effets d'obstruction semblent être les plus importants lorsque l'on tente de comprendre la diffusion du soluté. Alors que les effets de charge ne semblaient pas être importants pour l'autodiffusion de substrats chargés dans l'hydrogel d'alginate ou dans le biofilm bactérien, ils ont joué un rôle clé dans la compréhension de la diffusion à travers l’agarose. L’ensemble de ces résultats devraient être très utiles pour l'évaluation de la biodisponibilité des contaminants traces et des nanoparticules dans l'environnement. / Biofilms are primarily communities of microorganisms embedded in a complex exopolymer matrix. They are thought to play an important role as diffusive barriers in environmental systems and human health, resulting in increased resistance to disinfectants and antibiotics. Since mass transport in a biofilm is primarily due to molecular diffusion, it is critical to understand the main parameters influencing diffusive fluxes in a biofilm. In this thesis, a Pseudomonas fluorescens biofilm and two model hydrogels, (agarose and calcium alginate), were investigated. Both self-diffusion (Brownian motion) and mutual diffusion coefficients were quantified. Fluorescence correlation spectroscopy was used to measure the self-diffusion coefficients in a ca. 1 m3 confocal volume in the gels or biofilms, whereas a diffusion cell setup was employed for mutual diffusion measurements. In addition, microelectrode voltammetry was used to evaluate Donnan potential of the gels in order to determine its impact on diffusion. For the agarose hydrogel, the combined observations of a decreasing self-diffusion coefficient coupled with increasing mutual diffusion as a function of a decreasing ionic strength have been attributed to the gel’s Donnan potential. Measurements of the Donnan effect (difference of -30 mV between ionic strengths of 10-4 and 10-1 M) and the corresponding accumulation of ions in the hydrogel (13x enhancement with respect to the bulk solution) indicated that electrostatic interactions can strongly influence the diffusive flux of cations, even in a weakly charged hydrogel, such as agarose. Somewhat surprisingly, for a more highly charged gel such as calcium alginate, varying ionic strength and pH resulted in only small changes to the diffusion of charged probes in the hydrogel. These results suggested that the direct effect of the cations on gel structure (due to an induced swelling or compression) was much more effective than the Donnan effect when influencing solute diffusion. Similarly, for a bacterial biofilm, self-diffusion coefficients were virtually constant across a range of examined ionic strengths (10-4-10-1 M) for both negatively and positively charged small solutes (Db/Dw≈85%) and nanoparticles (Db/Dw≈50%), suggesting that the obstruction effect of the biofilms again overwhelmed the charge effect. The results of this work indicated that among the various major factors affecting diffusion in an oligotrophic environmental biofilm (steric exclusion, hydrophobic and electrostatic interactions), obstruction effects appeared to be the most important when attempting to understand the solute diffusion. While charge effects did not appear to be important to the self-diffusion of charged substrates in the alginate hydrogel or bacterial biofilm, they were key to understanding diffusion through another gel, with numerous biomedical and environmental applications, i.e. agarose. These results should be extremely useful when evaluating the bioavailability of the trace contaminants and nanoparticles in the environment.

Le diamant est un matériau fascinant avec d'exceptionnelles propriétés physiques. Son application à divers domaines reste limitée parce que sa fabrication est difficile et nécessite des substrats et conditions spécifiques. En outre, les dispositifs de diamant tels que les capteurs nécessitent généralement la structuration et l'échelle micro ou nanométrique, et l'inertie chimique du diamant rend ce processus technologique plus difficile que celui des semiconducteurs réguliers. Il s'agit d'un besoin évident de la recherche fondamentale d’explorer de nouvelles façons de fabriquer des nanostructures de diamant, ce qui permet de nouvelles formes de capteurs et dispositifs. Dans ce contexte, le travail présenté est d'une grande importance pour la communauté de diamant et pour le développement futur de la technologie du diamant.Le manuscrit est divisé en huit parties: une introduction; 6 chapitres, une conclusion générale. Dans l'introduction le contexte de l'étude est brièvement présenté avec les deux objectifs. Le premier consistait à étudier la croissance des nanofils de diamant et à trouver des conditions appropriées pour obtenir des nanofils de façon reproductible. Le deuxième objectif était la mise au point du procédé de gravure du diamant avec des particules de catalyseur et de l'optimisation des paramètres du procédé.Le premier chapitre de ce manuscrit présente tout d'abord l'état de l’art en mat ière de propriétés et des technologies de croissance du diamant. Puis, dans le deuxième chapitre, en vue de la croissance des nanofils et des études de gravure de nanostructures utilisées catalyseurs métalliques, la base de l'interaction métal-carbone est présenté.Le chapitre trois contient l'instrumentation et principe de fonctionnement des techniques expérimentales et analytiques utilisées dans cette étude. Le chapitre suivant se concentre sur la recherche de conditions favorables à la croissance des nanofils de diamant, d'abord en étudiant en détail un processus signalé en 2005 qui a conduit à la nucléation des nanocristaux sur des nanotubes de carbone, puis la croissance de nanofils.Les conditions de croissance ont été soigneusement reproduites, sans succès reproductible. Il en est déduit déduit que d'un élément non a contribué à la croissance, comme une contamination du catalyseur. La combinaison avec le fait que le processus publiée en 2005 n'a jamais été reproduite, en dépit de son importance technologique élevé, ce qui suggère que la contamination s'est produite également dans cette oeuvre originale.Puis, à partir de cette première observation, l'effet d'un catalyseur a été étudié, et des résultats intéressants ont été obtenus. Les nanofils ont été obtenus de façon reproductible, mais le point important est que les nanofils à base de silicium sont très faciles à cultiver, et qu'un environnement deCarbone pur était nécessaire d'étudier la croissance de nanofils de carbone. Dans ces conditions, un continuum allant de diamant de gravure pour la croissance du diamant a été obtenue en fonction de l'apport de carbone, très intéressant pour la technologie du diamant. Dans le cinquième chapitre du mécanisme de gravure de diamant par des particules de catalyseur est explorée. La gravure à motifs a été proposée pour la fabrication de nano-ou micro-structures dans le diamant, et il est présenté dans la dernière partie de ce chapitre. Le chapitre 6 présente deux applications intéressantes du processus dedéveloppement. Les premières membranes poreuses préoccupations utilisés comme bio-capteurs, et les nanotubes de carbone second concerne la base neuro-capteurs.Malgré l'étude infructueuse de la croissance du diamant nanofil, le travail fait des progrès significatifs à la science de la croissance matérielle nanocarbone. Et elle a conduit à l'étude approfondie de gravure diamant, qui est également très important pour la technologie. / One-dimensional structures with nanometre diameters, such as nanotubes and nanowires, have attracted extensive interest in recent years and form new family of materials that have characteristic of low weight with sometimes exceptional mechanical, electrical and thermal properties. Without any change in chemical composition, fundamental properties of bulk materials can be enhanced at the nanometre scale leading to extraordinary nanodevices.Since a few years, nanowires of different semiconducting materials have been grown. To mention few of these, Si, GaN, SnO, SiC and ZnO nanowires were all successfully demonstrated. However, the growth of diamond nanowires has not yet been demonstrated, despite the strong interest for this material. Bulk diamond combines various exceptional properties for a wide range of applications: Chemical inertness, radiation hardness, biocompatibility, high hole/electron mobility (2000/1000 cm2/V/s), high thermal conductivity (22 W/cm/K), wide bandgap (5.5 eV), and wide electric potential window (3.25 eV H-O evolutions).Since about 30 years, the growth of diamond thin film is well controlled either as insulator or as semiconductor with p- and n- type dopants. Fabrication of 25x25 mm2 monocrystalline diamond wafer has already been reported, and two inches wafers are expected in a couple of years demonstrating the growing interest for this material. Among present or short-term applications one can mention alpha-particle detectors, solar-blind UV sensors, high voltage electronic devices, bio-sensors and single photon source. The realization of nanowires should improve the performance of some of these devices and also open a range of new high performance applications.The stability of 0D (nanocrystals) and 1D (nanowires) diamond nanostructures has been extensively studied using ab initio modelling and indicates that for specific crystallographic orientations clusters of nanometric size are thermodynamically stable. One experimental indication for diamond nanowire growth has been published by Sun et al. in 2005, based on nanocrystal nucleation and growth on carbon nanotubes followed by 1D growth. This particular nucleation process on carbon nanotube has furthermore been explained theoretically in 2009.Based on these experimental and theoretical results, the first objective of this thesis was to explore the growth of diamond nanowire and find suitable conditions to obtain nanowires in a reproducible way. A wide range of process conditions were explored, first without any catalyst, then with metallic catalyst in order to promote Vapour-Liquid-Solid (VLS) growth. Although a comprehensive knowledge regarding carbon nanotube stability in hydrogen atmosphere and diamond-catalyst interaction has been obtained and some carbon nanostuctures were grown, no diamond nanowires were obtained in a reproducible way.However, the careful study of the diamond-catalyst interaction revealed a very interesting etching process that could be very useful for the fabrication of diamond nanostructures. A second objective was then defined: development of the etching process for diamond using transition metal as catalyst and optimization of the process parameters for specific applications such as the fabrication of porous diamond membranes for bio-sensors.

Submitted by Luciana Sebin (lusebin@ufscar.br) on 2016-10-05T18:08:34Z No. of bitstreams: 1 TeseERLR.pdf: 4461748 bytes, checksum: 2fe540767de5ff5f23af02775508026b (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-10-14T14:11:13Z (GMT) No. of bitstreams: 1 TeseERLR.pdf: 4461748 bytes, checksum: 2fe540767de5ff5f23af02775508026b (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-10-14T14:11:22Z (GMT) No. of bitstreams: 1 TeseERLR.pdf: 4461748 bytes, checksum: 2fe540767de5ff5f23af02775508026b (MD5) / Made available in DSpace on 2016-10-14T14:11:41Z (GMT). No. of bitstreams: 1 TeseERLR.pdf: 4461748 bytes, checksum: 2fe540767de5ff5f23af02775508026b (MD5) Previous issue date: 2015-12-15 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / This thesis combines image and signal processing to obtain virtual neuron distribution maps in a Microelectrode Array (MEA), which are devices designed for non-invasive electrophysiological signal recording for in vitro cultures of neuron cells. In the electrophysiological signal analysis, it is of interest the knowledge of the topological distribution of the cells along the MEA microelectrodes, but, usually the photographic images of the cell culture are not available. This doctoral work presents an approach to obtain the statistical topologic distribution of the neurons of an in vitro cell culture, denoted virtual distribution of neurons, from the electrophysiological signals. To certify that the statistical computation of the neuron counting is associated to each MEA microelectrode, it is used the ICA (Independent component Analysis) technique, for the separation of the neuron signals distributed throughout the MEA area, to obtain for each microelectrode, only the signals from its adjacent neurons. Assuming the hypothesis that the spontaneous neuron activities, spikes and bursts, are directly proportional to the neuron counting, it is realized the spike counting and burst counting, and it is assigned for each microelectrode, a number of neurons proportional to that numbers of activities. For the validation of the proposal, as well as for calibration of the system, to obtain the estimated number of neurons, it was used an experiment denoted 371, realized in Genoa University, Italy, in which it was recorded electrophysiological signals in 46 DIVs (Days In- Vitro), obtaining 20 minutes of recording in 25, 29, 32, 36, 39, 43, and 46 DIVs, and a set of photographic images in 38 DIV. Assuming that microelectrode neuron counting in the 38 DIV photographic image is proportional to the 39 DIV spontaneous electrophysiological activity signal recording, one day after the imaging, if was determined the neuron counting as function of the spontaneous electrophysiological activities recording, in a process denoted as calibration of the virtual number of neurons. The distance error from the neuron activities as function of the neuron counting in photographic image and in function of the recorded electrophysiological signals was calculated and compared for validation. In this way, it was possible to construct virtual topologic maps of neurons, proportional to the electrophysiological activities measured in 39 DIV, as a function of the spike and the burst countings. Comparing these two virtual maps, the spike counting virtual map was more close to the real neuron distributions viewed at the photographic image of 38 DIV. Also, the variance of the spike and burst counting along the 20 min of electrophysiological recording in a DIV, was calculated, and noted that the spike counting is more stable than burst counting. / Esta tese combina processamento de imagens e sinais, para a obtenção de uma distribuição virtual de neurônios em Matrizes de microeletrodos (Microelectrode Array, MEA), dispositivos projetados para o registro de sinais eletrofisiológicos de culturas de células neuronais, in-vitro, de forma não-invasiva. Na análise dos sinais eletrofisiológicos é de interesse o conhecimento da distribuição topológica das células ao longo dos microeletrodos, porém, nem sempre as imagens fotográficas das culturas são disponíveis. O presente trabalho apresenta uma metodologia de obtenção da distribuição topológica estatística dos neurônios numa cultura in-vitro, a partir dos sinais eletrofisiológicos. Para o cálculo estatístico do número de neurônios nessa distribuição topológica, é feito o uso da técnica de ICA (Independent Component Analysis), para obter os sinais relativos aos neurônios mais próximos para cada microeletrodo. Assumindo-se a hipótese de que as atividades eletrofisiológicas espontâneas dos neurônios, spikes e bursts, sejam diretamente proporcionais ao número de neurônios, realiza-se a contagem do número de spikes ou o número de bursts, e atribui-se o número de neurônios para cada microeletrodo, proporcionalmente à quantidade dessas atividades. Para a validação da proposta, foi utilizado um experimento, Experimento 371, realizado na Universidade de Gênova, Itália, em que foram registrados os sinais eletrofisiológicos ao longo de 46 DIVs (Dias In-Vitro), obtendo amostras de 20 minutos de registros para os 25, 29, 32, 36, 39, 43 e 46 DIVs, e um conjunto de imagens fotográficas da cultura no 38 DIV. Considerando-se que o número de neurônios associados a cada microeletrodo na imagem fotográfica no 38 DIV é proporcional à atividade eletrofisiológica espontânea dos neurônios, num registro realizado no 39 DIV, um dia após as fotos, foi feita uma regra de determinação do número virtual de neurônios em função das atividades eletrofisiológicas espontâneas medidas, denominada de calibração. O erro relativo à distância da atividade dos neurônios em relação à quantidade de neurônios na imagem fotográfica, e a atividade dos neurônios em função do registro de sinais eletrofisiológicos é calculado para comparação e validação. Dessa forma são construídos os mapas topológicos virtuais de neurônios proporcionais às atividades eletrofisiológicas medidas no 39 DIV, em função da quantidade de spikes e de bursts. O mapa obtido pela contagem de spikes se aproxima mais da distribuição real de neurônios vista na imagem fotográfica, do que o mapa obtido em função da contagem de bursts. No estudo de variância de atividades em função da contagem de spikes e bursts durante os 20 minutos de medidas num DIV, e constata-se que as atividades em contagem spikes é mais estável que em contagem de bursts.

Bacillus thuringiensis sérotype israelensis (Bti) produit quatre toxines entomocides utilisées à grande échelle pour le biocontrôle des populations de diptères nuisibles et vecteurs de maladies : Cry4Aa, Cry4Ba, Cry11Aa et Cyt1Aa. Chacune de ces toxines présente un effet létal sur différents insectes mais, lorsqu’elles sont combinées, on observe un effet synergique et l’absence de résistance. Bien que cette synergie soit bien documentée par des tests de toxicité, il existe très peu d’information sur son mécanisme aux niveaux cellulaire et moléculaire. À l’aide d’intestins isolés des larves du moustique Aedes aegypti, le principal vecteur du paludisme, et de microélectrodes, nous avons observé une dépolarisation membranaire en présence de Cyt1Aa et de Cry4Aa individuellement. Cette dépolarisation se produit cependant plus rapidement lorsque la Cyt1Aa est utilisée en même temps que la Cry4Aa. D’autre part, des expériences réalisées avec la sonde calcique Fura-2 sur une lignée cellulaire provenant d’Anopheles gambiae (Ag55), ont révélé une forte activité lytique de la Cyt1Aa, mais très peu d’effets des autres Cry, et ce même en combinaison. Nous avons dissocié les cellules de l’épithélium intestinal isolé du moustique pour des expériences de Fura2. Nos résultats, quoique préliminaires, montrent les effets variables de ces toxines lorsqu’elles sont administrées seules sur les cellules dissociées : une augmentation du calcium intracellulaire, ou une fuite de la sonde se traduisant par une perte du signal fluorescent, ou la lyse cellulaire. On observe également en présence de Cyt1Aa et de Cry4Ba, que les effets sont presque instantanés. / Bacillus thuringiensis var israelensis (Bti) produces four insecticidal toxins used around the world to control disease-borne and harmful dipterans populations: Cry4Aa, Cry4Ba, Cry11Aa and Cyt1Aa. They each present their lethal effect on different dipterans, but combined, they generate a synergistic activity and a reduced resistance is observed. Though these synergies are well documented and supported by toxicity bioassays, little is known regarding the cellular and molecular mechanisms of these synergies. Here, by using freshly isolated midguts from the mosquito Aedes aegypti, an important malaria vector, and glass microelectrodes, we measured the electrical potential of the apical membrane when exposed to these toxins alone or in combination. We observed a depolarisation when treated with Cyt1Aa and Cry4Aa. Toxin mixture assays only revealed a faster depolarisation of the membrane when the above two toxins were combined together, and a variety of responses with other toxin mixtures. Microspectrofluometry using the calcium probe Fura-2 on an immortal cell line from Anopheles gambiae (Ag55) showed massive effect of Cyt1Aa, but very little effect of the Cry toxins alone or in mixture. Microspectrofluometry experiments were also conducted on freshly dissociated cells from Aedes aegypti. Though these experiments are innovative and the results preliminary, it was observed that some cells responded differently to Cyt1Aa and Cry4Ba, showing the various ways these toxins affect cells, by inducing either intracellular calcium change, or by entirely losing the probe, or by cell lysis. The mixture of these toxins is very efficient and almost instantaneous.

This PhD research relates to the design, fabrication, characterization, and optimization of on-chip electrochemical microelectrode arrays (MEAs) for measurement of transient concentrations and gradients, focusing on fast transients and physiological applications. In particular, this work presents the determination of kinetic mechanisms taking place at an active interface (either physiological or non-physiological) in contact with a liquid phase using the MEA device to simultaneously estimate the concentration and gradient of the analyte of interest at the surface of the active interface. The design approach of the MEA device and the corresponding measurement methodology to acquire reliable concentration information is discussed. The ability of the MEA device to measure fast (i.e., in sub-second time scale) transient gradients is demonstrated experimentally using a controllable diffusion-reaction system which mimics the consumption of hydrogen peroxide by a 2D cell culture. The proposed MEA device and measurement methodology meet effectively most of the requirements for physiological applications and as a demonstration of this, two physiological applications are presented. In one application, the MEA device was tailored to measure the hydrogen peroxide uptake rate of human astrocytes and glioblastoma multiforme cells in 2D cell culture as a function of hydrogen peroxide concentration at the cell surface; the results allowed to quantitatively determine the uptake kinetics mechanisms which are well-described by linear and Michaelis-Menten expressions, in agreement with the literature. In the other application, further customization of the MEA device was realized to study the glucose uptake kinetics of human bronchial epithelial and small cell lung cancer cells, these latter with and without DDX5 gene knockdown; the results allowed to distinguish mechanistic differences in the glucose uptake kinetics among the three cell lines. These results were complemented with measurements of glycolytic and respiration rates to obtain a bigger picture of the glucose metabolism of the three cell lines. Finally, additional applications, both physiological and non-physiological, are proposed for the developed MEA device.

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