No / Aromatic aldehydes are good substrates of aldehyde dehydrogenase activity but are relatively poor substrates of aldehyde oxidase and xanthine oxidase. However, the oxidation of xenobiotic-derived aromatic aldehydes by thelatter enzymes has not been studied to any great extent. The present investigation compares the relative contribution of aldehyde dehydrogenase, aldehyde oxidase and xanthine oxidase activities in the oxidation of isovanillin in separate preparations and also in freshly prepared and cryopreserved liver slices. The oxidation of isovanillin was also examined in the presence of specific inhibitors of each oxidizing enzyme. Minimal transformation of isovanillin to isovanillic acid was observed in partially purified aldehyde oxidase, which is thought to be due to residual xanthine oxidase activity. Isovanillin was rapidly metabolized to isovanillic acid by high amounts of purified xanthine oxidase, but only low amounts are present in guinea pig liver fraction. Thus the contribution of xanthine oxidase to isovanillin oxidation in guinea pig is very low. In contrast, isovanillin was rapidly catalyzed to isovanillic acid by guinea pig liver aldehyde dehydrogenase activity. The inhibitor studies revealed that isovanillin was predominantly metabolized by aldehyde dehydrogenase activity. The oxidation of xenobiotic-derived aromatic aldehydes with freshly prepared or cryopreserved liver slices has not been previously reported. In freshly prepared liver slices, isovanillin was rapidly converted to isovanillic acid, whereas the conversion was very slow in cryopreserved liver slices due to low aldehyde dehydrogenase activity. The formation of isovanillic acid was not altered by allopurinol, but considerably inhibited by disulfiram. It is therefore concluded that isovanillin is predominantly metabolized by aldehyde dehydrogenase activity, with minimal contribution from either aldehyde oxidase or xanthine oxidase.

Cardiac tissue slices, a rarely used pseudo two-dimensional preparation, have gained increasing popularity for applications such as drug testing over the last ten years as they combine ease of handling with patho-physiologically relevant cell-type representation, distribution and inter-connection. The most well-established methods to measure electrophysiology in cardiac tissue are sharp electrodes and multi-electrode-arrays, techniques which are limited in spatial resolution or signal content. In this work, we have applied dual voltage Ca²⁺ optical mapping on cardiac slices, allowing us to record these two key parameters simultaneously at high spatio-temporal resolution, yielding better visualisation of conduction waves, spatial dispersion in action potential (AP) characteristics, and intracellular Ca²⁺ transient (CaT). The slice preparation method and the measurement protocols were refined to yield good reproducibility. Data analysis routines were developed to extract relevant parameters reliably. Despite being a promising candidate for drug testing, little is known about how slice and intact whole-heart AP properties are interrelated, and how to scale-up from observations in two dimensions (2D) to the three dimensional (3D) heart. In this thesis, we present a method to compare directly AP properties of intact whole-heart and tissue slices, and show the extent to which slices preserve AP characteristics. We have explored the suitability of tissue slices as an experimental model to study stretch induced changes in AP and CaT. During axial stretch, a dynamic profile of both AP and CaT was observed with an initial shortening of both AP and CaT duration, followed by a gradual recovery/prolongation. We have also used tissue slices to study spatial heterogeneity of AP and CaT properties in the rabbit left ventricular free wall. A transmural gradient can be captured in CaT and AP (with the longest APD and CaT durations being captured in the subendocardium). No large AP prolongation was found in the mid-myocardium. We conclude that the cardiac tissue slice preparation preserves some key functional parameters of the whole heart and is a promising model to study cardiac electrophysiology.

Mercury is an environmental contaminant that preferentially accumulates in the kidney. It has been previously shown using proton-induced X-ray emission analysis that mercury (HgCl2) accumulated in precision-cut rabbit renal cortical slices. In this study, the efficacy of seven chelating agents for the removal of Hg from renal dices has been examined. Rabbits were injected with HgCl2 (10 mg/kg) and 3 h later kidneys were sliced, or renal slices were exposed in vitro to a mildly toxic concentration of HgCl2 (5 x 10-5 M, 4 h). The slices were then treated in vitro with 10 mM concentrations of EDTA, lipoic acid (LA), penicillamine (PA), glutathione (GSH), 1,4-dithiothreitol (DTT), DMSA, or DMPS. DMPS proved to be the most effective in mobilizing Hg from in vivo or in vitro HgCl2-exposed renal tissue (>85% of control after 3 h incubation). Relative efficacies for the seven agents were DMPS > DMSA, PA > DTT, GSH > LA, EDTA. The use of renal slices appears to be a useful in vitro tool for assessing the efficacy of chelating agents on mobilizing accumulated Hg from renal tissue.

No description available.

A promising approach for the study of Glioblastoma are organotypic murine brain tissue slices as a substrate for the glioma cells to invade into. Current 3D assays based on this principle involved the use of tumor spheroids or cell suspensions in co-culture with the brain tissue slices. While this allowed for the study of glioma cell invasion, tumor spheroids lack the microarchitecture of patient-derived tumor tissue or glioma xenografts. This study has expanded this type of assay by investigating the viability of glioma xenograft slices in co-culture with organotypic murine brain tissue slices, proposing facile quantification methods of tumor growth and invasion and using this system for studying the effects of small molecule inhibitors. The organotypic murine brain tissue slices were prepared by slicing mouse brains in the coronal axis, using a vibratome, to a thickness of 300 µm. The slices were then transferred onto tissue culture membrane inserts for growth in air-liquid interface culture. A single mouse brain allowed for the production of multiple organotypic murine brain tissue slices, thus drastically reducing the number of animals needed for the study. GBM tumor xenografts from mice were sliced similarly on a vibratome, and circular portions with a diameter of 2 mm were placed on top of the murine brain tissue slices. After 7 days in culture, tissue slice co-cultures were analyzed by immunohistochemical staining of vertical sections, containing both the tumor and the murine brain tissue, for Type III intermediary filament proteins Vimentin and GFAP and the neural crest cell marker S-100. Independent of the cell line used for xenograft preparation, tumor tissues stained strongly positive for Vimentin, while the normal mouse brain tissue stained negative (in the studied region), so Vimentin was used as the primary tumor marker. For the quantification of the data acquired from micrographs, the tumor height, depth as well as the area of the invading cells and the tumor upper area (situated above the air margin of the brain tissue slice), the tumor lower area and the recipient tissue area were measured. From these parameters, a number of indices for each sample were derived, such as the tumor invasion index (TI-index), the tumor space occupying growth index (SOG-index), the tumor invasion depth index (TID-index), the space occupying growth depth index (SOGD-index) and the total tumor depth index (TTD-index). To validate the proposed quantification method, the results were compared with tumor spheroid tandem co-cultures. It was shown to be possible for GBM tumor xenografts to maintain their viability and invasive properties in co-culture with organotypic murine brain tissue slices. This was demonstrated immunohistochemically with xenografts from GBM cell lines G55T2, U-87 MG, LN-229 and T98G, displaying progressive tumor cell invasion from day 3 to 7 in co-culture. Tumor cell viability and proliferation in the ex vivo setting were also confirmed by Ki-67 staining. The usage of GBM tumor xenografts was also advantageous compared to spheroid-based assays. The direct comparison between the tumor spheroid and tumor xenograft co-cultures showed stark differences between assays even when using the same cell line. U-87 MG cells showed little or no invasiveness in the spheroid model but the glioma cells were diffusely spread into the murine brain tissue in the xenograft model. G55T2 xenograft co-cultures on the other hand displayed a significant increase of the SOG-index compared to their spheroid counterparts. Results were also compared to previous findings in an orthotopic tumor xenograft model, concluding that the proposed ex vivo model showed significant advantages compared to the orthotopic model by being more facile and cost-effective to implement and displaying comparatively more profound tumor invasiveness when studying the same cell lines. The strong increase of the invasive and space assuming properties of glioma cells in xenograft tissue slice tandem cultures also supports the hypothesis that they are superior to spheroid-based assays in studying tumor invasiveness. The developed GBM xenograft tissue slice tandem-cultures were also used for the ex vivo analysis of drug effects. The treatment with the Pim1 small molecule inhibitor SGI-1776 revealed after 7 days a decrease in the TI-index and SOG-index compared to the untreated group. A similar experiment was performed on spheroid co-cultures using a combination of SGI-1776 and the STAT3 inhibitor Stattic, also resulting in reduced TI- and SOG-indices. From this work, it can be concluded that the developed 3D ex vivo method is a facile and cost-effective platform to study the growth and invasiveness of GBM xenograft tumors in an in vivo-like environment. Owing to the large number of samples that can be generated from a single mouse, it has the potential to drastically reduce the number of animal experiments, addressing the 3R principle. It also showed more profound tumor cell invasiveness compared to spheroid-based ex vivo or orthotopic in vivo xenograft models and provides the quantification tools needed for preclinical drug testing. The model also has the potential to be expanded towards the usage of patient-derived tumor tissue as well as the preclinical testing of non-drug-based therapy options.:1 Introduction 1 1.1 Definition of Glioblastoma 1 1.2 Epidemiology, presentation 2 1.3 Etiology 5 1.4 Molecular pathways relevant in Glioblastoma tumorigenesis 5 1.5 Treatment options 9 1.6 Research models 11 1.7 Tissue slice models 15 1.8 Aims and research objectives 18 2 Publication 19 3 Summary 34 4 References 39 5 Supplementary materials 60 5.1 Additional materials 65 5.2 Comparison of immunohistochemical data to available literature 66 6 Darstellung des eigenen Beitrags 67 7 Erklärung über die eigenständige Abfassung der Arbeit 69 8 Publications 70 9 Curriculum vitae 71 10 Acknowledgements 72

Les cellules chromaffines des glandes surrénales sont des cellules neuroendocrines excitables impliquées dans la sécrétion de catécholamines. En réponse à un stress, ces hormones, parmi les premières à être libérées exercent de multiples actions sur leurs organes-cibles, contribuant à la réponse adaptive de l'organisme. Ainsi, élucider la physiopathologie du stress est un enjeu de santé publique et mieux connaître les mécanismes permettant au tissu médullosurrénalien d'optimiser la sécrétion de catécholamines aux besoins de l'organisme est un défi à relever.La sécrétion des catécholamines est liée à l'activité électrique des cellules chromaffines et élucider les mécanismes cellulaires qui en contrôlent l'excitabilité est d'intérêt. L'activité électrique de ces cellules est régulée par le nerf splanchnique ainsi que par des conductances ioniques intrinsèques. Dans ce contexte, les conductances opérant autour du potentiel de repos jouent un rôle majeur dans le déclenchement des potentiels d'action. C'est en particulier le cas du canal NALCN (sodium leak channel), récemment décrit comme régulant le potentiel de repos des neurones. C'est pourquoi nous avons orienté nos travaux vers la caractérisation du rôle de NALCN dans l'excitabilité des cellules chromaffines, dans des tranches de glandes surrénales de souris. L'enregistrement du potentiel de membrane révèle qu'environ 62% des cellules chromaffines présentent des potentiels d'action spontanés et que le profil de décharge suit un mode régulier ou un mode en bouffées. Des enregistrements plus longs révèlent qu'une même cellule présente alternativement ces 2 modes de décharge. Un changement de potentiel de quelques mV autour du potentiel de repos favorise un mode, indiquant que les courants ioniques actifs autour du potentiel de repos sont des composantes cruciales de l'excitabilité cellulaire. NALCN est-il un de ces courants?Pour commencer, nous avons observé, par hybridation in situ, la présence du transcrit codant NALCN dans les cellules chromaffines chez la souris (coll Dr. Ventéo, INM, Montpellier). Nous avons alors cherché à déterminer si NALCN est impliqué dans l'activité électrique des cellules chromaffines. Nous avons utilisé un protocole de diminution de la concentration extracellulaire de Na+, classiquement utilisé pour l'étude électrophysiologique de NALCN. La diminution du Na+ extracellulaire induit une hyperpolarisation et un arrêt des potentiels d'action. Cet effet n'est pas bloqué par la TTX. En potentiel imposé, la diminution du Na+ réduit le courant de maintien, elle n'est ni bloquée par la TTX ni par le Cs+. La courbe courant/potentiel du courant sensible à la réduction du Na+ révèle un courant linéaire entre -130 et -50 mV et un potentiel d'inversion en accord avec la contribution de plusieurs espèces ioniques. Ce courant présente une perméabilité majeure au Na+ vs K+. Ainsi, ces résultats décrivent une conductance ionique partageant des propriétés biophysiques et pharmacologiques similaires à celles de NALCN.Afin de poursuivre dans cette direction, nous avons initié des travaux ambitieux visant à éteindre l'expression du gène codant NALCN dans les cellules chromaffines, au travers d'une stratégie d'injection de virus in vivo. Une construction codant pour un shRNA dirigé contre NALCN, a été injectée dans la glande surrénale gauche. Les résultats sont très encourageants, montrant i) la présence, dans les glandes injectées, de cellules chromaffines transduites et ii) une diminution significative de l'expression de NALCN dans les glandes injectées avec le ShRNA-anti NALCN. Cette approche de transduction virale mérite d'être poursuivie.En conclusion, et même si les résultats actuels ne permettent pas d'affirmer avec certitude que NALCN contribue à l'excitabilité des cellules chromaffines, ce travail de thèse apporte néanmoins une contribution majeure à l'étude de l'excitabilité de ces cellules et ouvre des perspectives attractives quant au rôle de NALCN. / Adrenal chromaffin cells are excitable neuroendocrine cells involved in the secretion of catecholamines. Once delivered into the blood circulation, these hormones exert multiple actions, leading to physiological adjustments enabling the organism to cope with stress. Deciphering the physiology/pathology of stress is a major public health issue, especially in the field of the mechanisms that lead to optimal catecholamine secretion.The electrical activity of chromaffin cells critically shapes the catecholamine secretory pattern. Elucidating the mechanisms regulating the firing discharge is therefore of interest. In situ, chromaffin cell excitability is regulated by both the splanchnic nerve inputs and the intrinsic ion conductances expressed in cells. Regarding this, the conductances operating near the resting membrane potential are crucial in the cell competence to spontaneously fire. In particular, the background current flowing through the sodium leak channel NALCN has been recently reported to tune the resting potential of neuronal cells. This finding prompted us to investigate the possible contribution of NALCN to chromaffin cell excitability in mouse acute adrenal slices. The first part of my thesis was aimed at investigating chromaffin cell electrical firing pattern. Whole-cell recordings indicate that about 62% of mouse chromaffin cell spontaneously fire and exhibit two discharge patterns, a regular firing mode and a bursting mode. Long-lasting recordings of spontaneous electrical activity reveal that the two firing modes can occur in the same cells. When the membrane potential is challenged around the resting value, the firing pattern alternate between the two modes, indicating that currents operating around the resting membrane potential are key components in regulating cell excitability. Is NALN one of these currents?To answer this question, we first unveiled, by in situ hybridization, the presence of the transcript encoding NALCN in mouse chromaffin cells (coll with Dr. Ventéo, INM, Montpellier). Second, we performed electrophysiological experiments using protocols and pharmacological agents commonly used to study NALCN currents. Decreasing external NaCl leads to a robust membrane hyperpolarization, abrogating action potentials. This effect is not blocked by TTX. In voltage-clamp conditions, external Na+ reduction leads to a decrease in the holding current. This effect is not blocked by Cs+. Depolarizing voltage ramps unveil that the current blocked by lowering external Na+ blocks is linear between -130 and -50 mV, and displays a reversal potential arguing for a non-selective conductance. The ionic permeability is dominant for Na+ over K+. Collectively, our results describe a voltage-independent and non-selective cationic conductance operating near the resting potential of mouse chromaffin cells. Its electrophysiological and pharmacological properties recapitulate two NALCN attributes.In the third part, we developed an ambitious approach aiming at silencing NALCN expression specifically in chromaffin cells in vivo. Viral vectors encoding anti-NALCN shRNA under the control of the tyrosine hydroxylase promoter, as well as appropriate positive and negative viral constructs, were injected in the left gland. As promising results, transduced cells were detected in the injected glands only and a significant decrease in NALCN expression was observed in glands injected with the anti-NALCN shRNA. As such, the data collected from in vivo manipulation of NALCN expression are encouraging and this approach deserves to be pursued.This thesis describes a Na+-sensitive current operating near the resting membrane potential of mouse chromaffin cells, sharing biophysical and pharmacological properties with NALCN. Even though further experiments are needed to ascertain that NALCN supports this conductance, our work contributes to a better knowledge of chromaffin cell excitability.

With the advent of cloud computing and Software-as-a-Service, Service-Based Application (SBA) represents a new paradigm to build rapid, low-cost, interoperable and evolvable distributed applications. A new application is created by defining a workflow that coordinates a set of third-party Web services accessible over the Internet. In such distributed and loose coupling environment, the execution of SBA requires a high degree of flexibility. For example, suitable constituent services can be selected and integrated at runtime based on their Quality of Service (QoS); furthermore, the composition of service is required to be dynamically modified in response to unexpected runtime failures. In this context, the main objective of this dissertation is to design, to develop and to evaluate a service middleware for flexible execution of SBA by using chemical programming model. Using chemical metaphor, the service-based systems are modeled as distributed, selforganized and self-adaptive biochemical systems. Service discovery, selection, coordination and adaptation are expressed as a series of pervasive chemical reactions in the middleware, which are performed in a distributed, concurrent and autonomous way. Additionally, on the way to build flexible service based systems, we do not restrict our research only in investigating chemical-based solutions. In this context, the second objective of this thesis is to find out generic solutions, such as models and algorithms, to respond to some of the most challenging problems in flexible execution of SBAs. I have proposed a two-phase online prediction approach that is able to accurately make decisions to proactively execute adaptation plan before the failures actually occur.

This diploma thesis is focused on the evaluation of processed sliced products (cheese and analogues). For this purpose, was carried out sensory analysis of these products, a serial test and a questionnaire. Evaluation carried out a total of 65 assessors - 23 men and 42 women. The analysis of ranking showed that in the group of assessors was best evaluated processed wafer product (analogue) (average rank of 2,6) and processed sliced cheese (average rank of 2,7). For sensory analysis was found a significant role of fat on the dry basis of the product and the presence of added substances (extract from peppers, Emmental) on the perception of assessors. The survey was focused on the preference of the attractiveness of packaging of selected processed cheese slices and sliced processed products, furthermore focused on understanding the concept of the analogue product. Assessors have demonstrated their knowledge of the concept of analogue product only in 14% of cases.

Orientador: Luiz Felipe Mendes de Moura / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Mecânica / Made available in DSpace on 2018-08-16T08:00:05Z (GMT). No. of bitstreams: 1 Rodrigues_AlexCristiano_M.pdf: 7859697 bytes, checksum: f1415c4744eaf2e3068a564f5afe9f1c (MD5) Previous issue date: 2009 / Resumo: Este trabalho apresenta a utilização de um modelo numérico para a determinação das forças hidrodinâmicas atuantes em comportas planas tipo vagão, utilizadas em obras de desvio de Usinas Hidrelétricas. Para isso foi utilizado o software comercial Fluent, onde foi gerado um modelo numérico tridimensional para diversas aberturas da comporta vagão ensecadeira, a qual é responsável pelo fechamento em definitivo do desvio. A validação dos resultados numéricos foi realizada através da comparação de dados de um modelo físico reduzido previamente estudado em laboratório, onde constatou-se a correta captação e interpretação dos fenômenos hidráulicos presentes, permitindo utilizar sua metodologia para aplicações em obras semelhantes. A vazão e seu respectivo coeficiente de descarga apresentaram diferenças médias da ordem de 8%, as pressões atuantes no fundo do canal apresentam diferenças máximas de 3,6%, enquanto que os esforços hidrodinâmicos registraram diferenças médias de 7%. As maiores variações foram observadas nas menores aberturas, quando a vazão é determinada pela comporta, e as velocidades são mais altas. A título de comparação dos fenômenos hidráulicos, foram levantados os dados pertinentes a outros três ensaios físicos de túneis de desvio. Estes resultados foram comparados com o ensaio numérico e a formulação teórica, indicando que esta última, de uma forma geral, possui resultados mais conservadores / Abstract: This work presents a proposal of a numerical model to determine the hydraulic forces acting in fixed wheel gates utilized in hydroelectric power plants. Fluent commercial CFD (computational fluid dynamics) software was used to generate a three-dimensional model of the hydraulic gate; different models were generated for different opening positions of the hydraulic gate. The results obtained from the numerical model were validated by comparing them to results obtained from a reduced model previously built in a hydraulic laboratory. When comparing these results it was possible to realize that the numerical model could well represent the hydraulic effects, allowing possibility of the methodology application for similar works. The volume flow and discharge coefficients presented a average variation of 8%; pressure distribution at the bottom of the canal presented a average variation of 3,6%; and hydraulic forces presented an average of 7% difference. Higher differences occurred at lowest openings, when the flow is mostly controlled by the gate and speed is higher. As a complement to the work, results from three other laboratory reduced models were compared to analytical proposals found in literature and numerical simulation results. These comparisons show that these analytical models are usually conservative / Mestrado / Termica e Fluidos / Mestre em Engenharia Mecânica

Single photon emission tomography (SPECT) data from myocardial perfusion imaging (MPI) are normally displayed as a set of three slices orthogonal to the left ventricular (LV) long axis for both ECG-gated (GSPECT) and non-gated SPECT studies. The total number of slices presented for assessment depends on the size of the heart, but is typically in excess of 30.  A requirement for data presentation is that images should be orientated about the LV axis; therefore, a set of radial slice would fulfill this need. Radial slices are parallel to the LV long axis and arranged diametrically. They could provide a suitable alternative to standard orthogonal slices, with the advantage of requiring far fewer slices to adequately represent the data. In this study a semi-automatic method was developed for displaying MPI SPECT data as a set of radial slices orientated about the LV axis, with the aim of reducing the number of slices viewed, without loss of information and independent on the size of the heart. Input volume data consisted of standard short axis slices orientated perpendicular to the LV axis chosen at the time of reconstruction.  The true LV axis was determined by first determining the boundary on a central long axis slice, the axis being in the direction of the y-axis in the matrix. The skeleton of the myocardium were found and the true LV axis determined for that slice. The angle of this axis with respect to the y-axis was calculated. The process was repeated for an orthogonal long axis slice. The input volume was then rotated by the angles calculated. Radial slices generated for presentation were integrated over a sector equivalent to the imaging resolution (1.2 cm); assuming the diameter of the heart is about 8cm then non-gated data could be represented by 20 radial slices integrated over an 18 degree section. Gated information could be represented with four slices spaced at 45 intervals, integrated over a 30 degree sector.