Foram conduzidos experimentos com o objetivo de avaliar a bioatividade e o modo de ação de extratos aquosos e orgânicos de ramos de Trichilia pallida Swartz e extratos aquosos de sementes de nim, Azadirachta indica A. Juss, em relação a ninfas e ovos da mosca-branca Bemisia tabaci (Genn.) biótipo B. Inicialmente, verificou-se que o extrato aquoso de sementes de nim apresenta ação inseticida sobre ninfas de mosca-branca pelas vias translaminar, sistêmica e de contato, nas concentrações de 1; 0,5 e 0,3% (p/v), respectivamente. Em seguida, na comparação entre os extratos metanólico, etanólico, clorofórmico e hexânico de ramos de T. pallida, na concentração de 5% (p/v), foi selecionado o extrato clorofórmico como mais eficiente sobre ninfas de mosca-branca. Esse extrato foi comparado com os extratos aquosos de sementes de nim e de ramos de T. pallida sobre as ninfas quanto aos três tipos de ação nas respectivas concentrações determinadas no experimento. Apenas o extrato de nim provocou mortalidade ninfal pelas três vias testadas. Em relação à fase de ovo, constatou-se que a sua idade não afeta a ação ovicida do extrato aquoso de sementes de nim. Na comparação da ação translaminar e sistêmica dos extratos aquoso e clorofórmico de ramos de T. pallida e aquoso de sementes de nim, a 3% (p/v), sobre os ovos de mosca-branca, verificou-se que apenas o extrato de nim tem efeito ovicida. / The objective of this research was to determinate the bioactivity and the action means of aqueous and organic extracts of Trichilia pallida Swartz twigs, and the aqueous extracts of neem seeds, Azadiracta indica A. Juss, on nymphs and eggs of silverleaf whitefly. Initially, it was observed that aqueous extracts of neem seeds caused insecticidal activity on the nymphs by translaminar (1%), systemic (0,5%) and topical action (0,3%). After, in the comparison of metanolic, etanolic, chloroformic, and hexanic T. pallida twig extracts at the concentrations of 5%, the chloroformic extract was more efficient on the nymphs. This extract was compared with aqueous extracts of neem seeds, and the T. pallida twigs, by three means (translaminar, systemic and topical action) at concentrations previously determinated. Just the neem extract caused nymphal mortality for three means. In relation to the phase of eggs, it was verified that the age did not affect the ovicidal action of aqueous extract of neem seeds. In the comparison of translaminar and sistemic action of aqueous and chloroformic extracts of T. pallida twigs and aqueous extracts of neem seeds (3%) on eggs of the insect, just the neem extract showed ovicidal activity.

Em 1903, cerca de duzentos representantes das classes produtoras reuniram-se em Belo Horizonte para a realização do Congresso Agrícola, Industrial e Comercial de Minas Gerais. Estes homens foram convidados a debater medidas para alavancar o progresso do estado, que enfrentava forte crise econômica, decorrente, sobretudo, da queda nos valores do café, seu principal produto de exportação. O Congresso nasceu de uma iniciativa do presidente do estado, Francisco Salles, encabeçada por João Pinheiro da Silva, cujo intuito era ouvir representantes das atividades produtivas. Vindos de diversas regiões, principalmente do Centro, da Mata e do Sul, eles construíram um amplo painel da economia mineira, no qual inseriram questões sobre a educação. Nesta tese, tendo como principais fontes periódicos, legislação e biografias, procurou-se analisar essas manifestações em conjunto e separadamente, identificando autores, objetivos, destinatários e referências para a compreensão do que, na passagem do século XIX para o XX, deveria ser o ensino profissional ou, precisamente, a educação considerada como necessária e propícia ao progresso econômico. O representantes das classes produtoras apresentaram propostas de instrução com enfoques diferenciados com relação ao tipo de ensino, suas modalidades e níveis. Também foi possível identificar aspectos comuns ao debate educacional coetâneo, tais como a atribuição do atraso econômico à falta de instrução que preparasse para o trabalho. Desse modo, os representantes apontaram a necessidade de modificar técnicas, procedimentos, instrumentos e materiais para uma produção mais moderna e racional, e defenderam propostas de formação dos dirigentes das atividades produtivas e dos quadros técnicos e a educação do trabalhador. / In 1903, about two hundred representatives of the producing classes met in Belo Horizonte for the achievement of the Agricultural, Industrial and Commercial Congress of Minas Gerais. These men were invited to discuss means to leverage the state progress, which faced severe economic crisis, chiefly due to the fall in coffee values, its main export product. The congress was an initiative of the president of the state, Francisco Salles, headed by João Pinheiro da Silva, and aimed to hear representatives of productive activities. Coming from various regions, particularly the Centre, the Mata and the South, they built a broad panel of Minas Gerais economy, in which they introduced questions about education. In this thesis, using mainly journalistic sources, legislation and biographies, we sought to analyze these events together and separately, identifying authors, objectives, receivers and references to the understanding of what should be vocational education, namely the education considered necessary and propitious to economic progress in the late nineteenth century to the twentieth. The representatives of the producing classes presented instruction propositions with different approaches regarding the type of school, its modalities and levels. It was also possible to identify common aspects of coeval educational debate, such as the association of economic backwardness to the lack of instruction to prepare for work. Thus, the representatives pointed out the need to modify techniques, procedures, tools and materials for a more modern and rational production and advocated propositions on the formation of leaders of productive activities and technical staff and on the worker education.

Type I diabetes (T1D) is caused by autoimmune destruction of pancreatic β-cells. Immediate consequences of T1D are severe weight loss, ketoacidosis and death unless insulin is administered. The long-term consequences of T1D are dysregulation of metabolism leading to cardiovascular complications, neuropathy and kidney insufficiency. It is estimated that 3 million Americans have T1D, and its prevalence among young individuals is progressively rising. Islet transplantation is the most effective way to treat T1D. Unfortunately, there is a chronic shortage of cadaveric organ donors to treat all of the patients on the waiting list. Thus, an alternative source of insulin producing cells (IPCs) could significantly improve patient treatment. Our lab seeks to establish human induced pluripotent stem (iPS) cells as a novel source of IPCs that are patient tailored. The aim of this thesis was to 1) compare the differentiation of T1D and nondiabetic (ND) patient-derived iPS cells into IPCs, and 2) devise an effective protocol for differentiating skin fibroblast-derived T1D iPS cells into functional, glucose-responsive IPCs. Initially, T1D iPS cells were differentiated into IPCs. However, the yield was very poor. We hypothesized that epigenetic barriers were prevalent in T1D iPS cells, limiting their differentiation into IPCs. To address this problem, we utilized 5-aza-2’-deoxycytidine (5-aza-DC), a potent demethylating agent that inhibits the DNA methyltransferase (Dnmt). We reasoned that the use of a demethylation agent might induce a more labile, permissive state, allowing for greater cell responses to differentiation stimuli. Typically, after the differentiation of T1D iPS cells, several cell cluster types are obtained, namely compact cell clusters and hollow cysts. 5-aza-DC treatment appeared to convert all of the cell clusters into characteristic islet-like compact structures. In contrast, in untreated T1D IPC cultures, we observed the dominant presence of many hollow cysts with only a few tight spheroids. The hollow cysts stained negative for insulin whereas the rare solid spheroids highly expressed insulin. Flow cytometry analysis indicated a much greater percentage of Pdx1+ and insulin+ cells in 5-Aza-DC-treated cultures. These cells express markers typical of pancreatic β-cells, possessed insulin granules in similar quantities as islets, and were glucose-responsive. When transplanted in immunodeficient mice that had developed streptozotozin-induced diabetes, there was a dramatic decrease of hyperglycemia within 28 days. These mice effectively managed glucose challenge by recovering to normoglycemia, whereas nontransplanted mice did not. Altogether, our data for the first time reveal a very high yield of functional IPCs derived from human iPS cells derived from a patient with T1D, which presents a novel alternative source of IPCs that could be used to treat T1D.

Recursion is a fundamental tool of mathematics used to define, construct, and analyze mathematical objects. This work employs induction, sieving, inversion, and other recursive methods to solve a variety of problems in the areas of algebraic number theory, topological and combinatorial graph theory, and analytic probability and statistics. A common theme of recursively defined functions, weighted sums, and cross-referencing sequences arises in all three contexts, and supplemented by sieving methods, generating functions, asymptotics, and heuristic algorithms. In the area of number theory, this work generalizes the sieve of Eratosthenes to a sequence of polynomial values called polynomial-value sieving. In the case of quadratics, the method of polynomial-value sieving may be characterized briefly as a product presentation of two binary quadratic forms. Polynomials for which the polynomial-value sieving yields all possible integer factorizations of the polynomial values are called recursively-factorable. The Euler and Legendre prime producing polynomials of the form n2+n+p and 2n2+p, respectively, and Landau's n2+1 are shown to be recursively-factorable. Integer factorizations realized by the polynomial-value sieving method, applied to quadratic functions, are in direct correspondence with the lattice point solutions (X,Y) of the conic sections aX2+bXY +cY2+X-nY=0. The factorization structure of the underlying quadratic polynomial is shown to have geometric properties in the space of the associated lattice point solutions of these conic sections. In the area of combinatorial graph theory, this work considers two topological structures that are used to model the process of homologous genetic recombination: assembly graphs and chord diagrams. The result of a homologous recombination can be recorded as a sequence of signed permutations called a micronuclear arrangement. In the assembly graph model, each micronuclear arrangement corresponds to a directed Hamiltonian polygonal path within a directed assembly graph. Starting from a given assembly graph, we construct all the associated micronuclear arrangements. Another way of modeling genetic rearrangement is to represent precursor and product genes as a sequence of blocks which form arcs of a circle. Associating matching blocks in the precursor and product gene with chords produces a chord diagram. The braid index of a chord diagram can be used to measure the scope of interaction between the crossings of the chords. We augment the brute force algorithm for computing the braid index to utilize a divide and conquer strategy. Both assembly graphs and chord diagrams are closely associated with double occurrence words, so we classify and enumerate the double occurrence words based on several notions of irreducibility. In the area of analytic probability, moments abstractly describe the shape of a probability distribution. Over the years, numerous varieties of moments such as central moments, factorial moments, and cumulants have been developed to assist in statistical analysis. We use inversion formulas to compute high order moments of various types for common probability distributions, and show how the successive ratios of moments can be used for distribution and parameter fitting. We consider examples for both simulated binomial data and the probability distribution affiliated with the braid index counting sequence. Finally we consider a sequence of multiparameter binomial sums which shares similar properties with the moment sequences generated by the binomial and beta-binomial distributions. This sequence of sums behaves asymptotically like the high order moments of the beta distribution, and has completely monotonic properties.

Slow-release devices retained in the rumen, are a simple method for continuous administration of bioactives to ruminant animals. To satisfy regulatory requirements and avoid waste of bioactive due to under- or over-dosing, it is advantageous to have a constant and predictable release rate. Existing intraruminal controlled-release technologies cannot easily be adapted for different bioactives or rates of release and can be influenced by the variable physiological environment in the rumen. Some existing commercial products use the pressure generated by a hydrogen gas-producing cell to extrude fluids from a syringe-like device. This technology may provide advantages for ruminal controlled-release as the gas production rate is unaffected by environment in the rumen and can be easily adjusted using electrical resistance applied to the gas cell. This technology was adapted for use in the rumen in these studies. Initial experiments identified the need for greater understanding of the rate that hydrogen is produced by the gas cell and the rate that gas diffuses through the barrel walls. Gas production rate was found to be inversely proportional to the resistance applied to the gas-producing cell. Factors affecting gas diffusion rate from the device were studied and a polymer was identified that reduced hydrogen diffusion to 5% of that for the initial components used. A relationship was developed to predict the release profile of a device. Controlled-release devices were constructed from selected materials. They released blank formulation at in vitro at a constant rate, which was within experimental variation of predicted values. Release rates from the devices used in vivo were slightly higher than predicted. The presence of rumen gases inside in vivo devices suggested that the difference may be due to inward diffusion of these gases; these may be eliminated by further study of barrel materials. Recommendations on the redesign of this technology for use as a generic intraruminal delivery system are given.

<p>Vaccination with DNA encoding the encephalitogenic autoantigen myelin oligodendrocyte glycoprotein (MOG), pMOG91-108, induce a protective immunity against experimental autoimmune encephalomyelitis (EAE), an animal model of human multiple sclerosis. By injection of a DNA vaccine that contains a DNA region encoding short interfering RNA specific for IFNβ (pMOG-IFNβ) the protective effect of the DNA vaccination is totally inhibited. This demonstrates that IFN-β is directly involved in the protective mechanism against EAE.</p><p>The objective of this project was to study how molecules involved in the inflammatory process in EAE are regulated by suppressive DNA vaccination. mRNA expression of IL-1β, TGF β, IL-23p40 and Axl receptor tyrosine kinas did not show any significant differences between the groups vaccinated with these DNA vaccines. IL-6 and IFNγ mRNA expression after MOG stimulation in rats treated with pCI, a control vaccine was significantly higher compared to the group vaccinated with vaccine containing pMOG-IFNβ. IL-17 m RNA expression after MOG stimulation in pCl-treated rats was significantly higher compared to the group vaccinated with vaccine containing pMOG-91-108. Of these results the mRNA expression of IL-17 and IL-6 were of interest for the project.</p><p>The immune system normally protects the body against infections and T-cells have an important role in this defence system. In MS and EAE, the immune system attacks the myelin and this process is caused by a dysregulation of the T-cells. IL-17-producing Th17 cells mediate EAE. Naïve CD4 T-cells in the presence of IL-6 and TGFβ are differentiated to Th17 cells instead of differentiating into T-helper or regulatory T-cells. These IL-17-producing T-cells are highly pathogenic and essential for the development of EAE. The results showed that pMOG IFNβ vaccine had an effect at the immune response, which resulted in an inhibition of the IL-6 production and that vaccination with pMOG91-108 impairs differentiation of IL-17-producing T-cells.</p>

Vaccination with DNA encoding the encephalitogenic autoantigen myelin oligodendrocyte glycoprotein (MOG), pMOG91-108, induce a protective immunity against experimental autoimmune encephalomyelitis (EAE), an animal model of human multiple sclerosis. By injection of a DNA vaccine that contains a DNA region encoding short interfering RNA specific for IFNβ (pMOG-IFNβ) the protective effect of the DNA vaccination is totally inhibited. This demonstrates that IFN-β is directly involved in the protective mechanism against EAE. The objective of this project was to study how molecules involved in the inflammatory process in EAE are regulated by suppressive DNA vaccination. mRNA expression of IL-1β, TGF β, IL-23p40 and Axl receptor tyrosine kinas did not show any significant differences between the groups vaccinated with these DNA vaccines. IL-6 and IFNγ mRNA expression after MOG stimulation in rats treated with pCI, a control vaccine was significantly higher compared to the group vaccinated with vaccine containing pMOG-IFNβ. IL-17 m RNA expression after MOG stimulation in pCl-treated rats was significantly higher compared to the group vaccinated with vaccine containing pMOG-91-108. Of these results the mRNA expression of IL-17 and IL-6 were of interest for the project. The immune system normally protects the body against infections and T-cells have an important role in this defence system. In MS and EAE, the immune system attacks the myelin and this process is caused by a dysregulation of the T-cells. IL-17-producing Th17 cells mediate EAE. Naïve CD4 T-cells in the presence of IL-6 and TGFβ are differentiated to Th17 cells instead of differentiating into T-helper or regulatory T-cells. These IL-17-producing T-cells are highly pathogenic and essential for the development of EAE. The results showed that pMOG IFNβ vaccine had an effect at the immune response, which resulted in an inhibition of the IL-6 production and that vaccination with pMOG91-108 impairs differentiation of IL-17-producing T-cells.

Improving methods of hydrocarbon production and developing new techniques for the creation of natural gas storage facilities are critically important for the petroleum industry. This dissertation focuses on two key topics: (1) mechanisms of sand production from petroleum reservoirs and (2) mechanical characterization of caverns created in carbonate rock formations for natural gas storage. Sand production is the migration of solid particles together with the hydrocarbons when extracted from petroleum reservoirs. It usually occurs from wells in sandstone formations that fail in response to stress changes caused by hydrocarbon withdrawal. Sand production is generally undesirable since it causes a variety of problems ranging from significant safety risks during high-rate gas production, to the erosion of downhole equipment and surface facilities. It is widely accepted that a better understanding of the mechanics of poorly-consolidated formations is required to manage sand production; which, in turn, enables the cost effective production of gas and oil resources. In this work, a series of large-scale laboratory experiments was conducted in fully saturated, cohesionless sand layers to model the behavior of a petroleum reservoir near a wellbore. We directly observed several key characteristics of the sand production phenomenon including the formations of a stable cavity around the wellbore and a sub-radial flow channel at the upper surface of the tested layer. The flow channel is a first-order feature that appears to be a major part of the sand production mechanism. The channel cross section is orders of magnitude larger than the particle size, and once formed, the channel becomes the dominant conduit for fluid flow and particle transport. The flow channel developed in all of our experiments, and in all experiments, sand production continued from the developing channel after the cavity around the borehole stabilized. Our laboratory results constitute a well constrained data set that can be used to test and calibrate numerical models employed by the petroleum industry for predicting the sand production phenomenon. Although important for practical applications, real field cases are typically much less constrained. We used scaling considerations to develop a simple analytical model, constrained by our experimental results. We also simulated the behavior of a sand layer around a wellbore using two- and three-dimensional discrete element methods. It appears that the main sand production features observed in the laboratory experiments, can indeed be reproduced by means of discrete element modeling. Numerical results indicate that the cavity surface of repose is a key factor in the sand production mechanism. In particular, the sand particles on this surface are not significantly constrained. This lack of confinement reduces the flow velocity required to remove a particle, by many orders of magnitude. Also, the mechanism of channel development in the upper fraction of the sample can be attributed to subsidence of the formation due to lateral extension when an unconstrained cavity slope appears near the wellbore. This is substantiated by the erosion process and continued production of particles from the flow channel. The notion of the existence of this surface channel has the potential to scale up to natural reservoirs and can give insights into real-world sand production issues. It indicates a mechanism explaining why the production of particles does not cease in many petroleum reservoirs. Although the radial character of the fluid flow eventually stops sand production from the cavity near the wellbore, the production of particles still may continue from the propagating surface (interface) flow channel. The second topic of the thesis addresses factors affecting the geometry and, hence, the mechanical stability of caverns excavated in carbonate rock formations for natural gas storage. Storage facilities are required to store gas when supply exceeds demand during the winter months. In many places (such as New England or the Great Lakes region) where no salt domes are available to create gas storage caverns, it is possible to create cavities in limestone employing the acid injection method. In this method, carbonate rock is dissolved, while CO₂ and calcium chloride brine appear as products of the carbonate dissolution reactions. Driven by the density difference, CO₂ rises towards the ceiling whereas the brine sinks to the bottom of the cavern. A zone of mixed CO₂ , acid, and brine forms near the source of acid injection, whereas the brine sinks to the bottom of the cavern. Characterization of the cavern shape is required to understand stress changes during the cavity excavation, which can destabilize the cavern. It is also important to determine the location of the mixture-brine interface to select the place of acid injection. In this work, we propose to characterize the geometry of the cavern and the location of the mixture-brine interface by generating pressure waves in a pipe extending into the cavern, and measuring the reflected waves at various locations in another adjacent pipe. Conventional governing equations describe fluid transients in pipes loaded only by internal pressure (such as in the water hammer effect). To model the pressure wave propagation for realistic geometries, we derived new governing equations for pressure transients in pipes subjected to changes in both internal and external (confining) pressures. This is important because the internal pressure (used in the measurement) is changing in response to the perturbation of the external pressure when the pipe is contained in the cavern filled with fluids. If the pressure in the cavern is perturbed, the perturbation creates an internal pressure wave in the submerged pipe that has a signature of the cavern geometry. We showed that the classic equations are included in our formulation as a particular case, but they have limited validity for some practically important combinations of the controlling parameters. We linearized the governing equations and formulated appropriate boundary and initial conditions. Using a finite element method, we solved the obtained boundary value problem for a system of pipes and a cavern filled with various characteristic fluids such as aqueous acid, calcium chloride brine, and supercritical CO₂ . We found that the pressure waves of moderate amplitudes would create measurable pressure pulses in the submerged pipe. Furthermore, we determined the wavelengths required for resolving the cavern diameter from the pressure history. Our results suggest that the pressure transients technique can indeed be used for characterizing the geometry of gas storage caverns and locations of fluid interfaces in the acid injection method.

[pt] Quando um poço de petróleo é completado em arenito brando e pobremente consolidado é possível que ocorra fluxo de areia para dentro do poço. Problemas derivados da produção de areia incluem a obstrução do poço, abrasão de revestimentos e ferramentas, colapso do revestimento e necessidade de separação e disposição, em superfície, de areias impregnadas por óleo. Contudo, em reservatórios de óleo pesado, a alta viscosidade e abaixa produtividade significam que os problemas mencionados acima são menos críticos. Nestes casos, alguma produção de areia é aceitável para aumentar a produtividade.A produção de areia é notória em arenitos mal consolidados, mas também podem ocorrer em arenitos coesivos e medianamente resistentes como resultado da perturbação imposta à rocha durante as operações de perfuração, completação e produção Provavelmente o principal efeito é a destruição da cimentação mineral que produz uma região com material granular na vizinhança do poço. As forças de percolação criadas pelo fluxo podem então arrastar as partículas sólidas para o interior do poço.Um modelo contitutivo que seja capaz de reproduzir o comportamento tensão-deformaçãoresistência de arenitos é de grande interesse para estudos da previsão da produção de areia. Este tema é o ponto central desta tese.Uma revisão dos artigos relacionados com os mecanismos de produção de areia é apresentado no capítulo 2.Dois arenitos brandos obtidos de afloramentos foram caracterizados. Após a diagnose petrográfica realizou-se o tratamento e processamento de imagens da lâminas para mensuração da distribuição granulométrica, parâmetros texturais e dimensão dos poros. Os argilominerais foram investigados por difração de raios- X, usando quatro tipos distintos de preparação de amostras. A porosidade e a permeabilidade intrínseca foram determinadas usando um porosímetro de Boyle e um permeametro à gás. Empregou-se um algoritmo para análise multivariada dos dados para distinguir entre grupos de arenitos com propriedades físicas semelhantes.Realizou-se um exercício numérico, usando soluções analíticas para a distribuição das tensões na parede de um poço, com o objetivo de se identificar as possíveis trajetórias de tensão que ocorrem, considerando as tensões in situ, a perfuração e a produção de fluidos.Foi desenvolvido um programa experimental de ensaios mecânicos que inclui testes uniaxiais,triaxiais convencionais e hidrostático, além de carregamentos seguindo a trajetória de p constante. Estes ensaios serviram para obtenção dos parâmetros do modelo de Lade, bem como para caracterizar o comportamento geomecânico dos arenitos. Deve-se mencionar que os corpos de prova estavam totalmente saturados com água e com óleo. Usou-se um algoritmo de otimização para identificação dos parâmetros do modelo. Por fim, avaliou-se a capacidade do modelo elastoplástico de Lade & Kim para descrever o comportamento tensão deformação dos arenitos estudados. / [en] When a well completed in a poorly consolidated and weak sandstone is produced, sand inflow from reservoir can occour. Problems arising from sand production include sand bridging and wellbore plugging; tools and casing abrasion requiring frequent workovers; casing collapse and distortion in the production zone; sand separation at surface and disposal of an environmentaly contaminated sand. However in heavy oil reservoirs, high viscosity and low production rates means that the problems mentioned before are far less than in high rate light oil wells. In these cases, some sand production can be accepted in order to increase productivity. Sand production is a conspicuous phenomena in poorly consolidated sandstones, but can also occur in cohesive medium-strong sandstones as a result of rock fabric damage during drilling, completion and production activities. Probably the main disturbance imposed to rock is the breakage of mineral cement, which produces a region of a granular yielded material around the bore. The percolation forces created by fluid flow can drag the solid particles to the well. A constitutive model that can be able to simulate stress-strain behavior is of major interest to sand production prediction. This theme is the central point of this work. A review of published articles related to sand production mechanisms is presented in the second chapter. Two weak sandstones obtained from outcrops were characterized. After a diagnosis under petrographic thin sections, an extensive image treatment and processing were carried out to measure grain size distribution, textural parameters and pore dimensions. Clay mineral were investigated by mean of X-ray diffraction techniques, using four distinct kinds of sample preparation. Porosity and intrinsic permeability were determined using a Boyle s principle porosimeter and a gas permeameter. A multivariate data analysis (cluster analysis) algorithm based on distance measures were applied to distinguish among groups with similar physical properties. A numerical exercise using analytical solution equations for the stress distribution on the wall of the well was done to determine the stress path, considering drilling and production effects. A experimental program of rock mechanic tests including uniaxial, conventional triaxial, hydrostatic and proportional loading, besides tests following a (p) constant path, were run in order to characterize the rock behavior as well as to obtain the constitutive model parameters. It must be mentioned that samples were water and oil saturated. An optimization algorithm for of model parameters identification were used and the Lade & Kim elastoplastic constitutive model was evaluated for describe stress-strain relation of sandstones.

Primary aldosteronism is the most common secondary cause of hypertension with a prevalence of about 10%. About half of PA cases are caused by aldosterone-producing adenomas (APA). Two APA subtypes, ZG-like and ZF-like APAs, have been described, according to the histological resemblance to normal zona glomerulosa (ZG) and zona fasciculata (ZF), underlying somatic mutations (KCNJ5 commonly found in ZF-like, CACN1AD, ATP1A1, ATP2B3, CTNNB1 in ZG-like APAs), and transcriptome profile. It is unknown if the process of tumorigenesis differs between ZG- and ZF-like APAs. In order to define ZG specific genes, we have compared the transcriptome of APAs and their adjacent adrenal glands by microarray assay. RNA was isolated by laser capture microdissection (LCM) from adjacent ZG, ZF and APAs from 14 patients with Conn’s and 7 patients with phaeocromocytoma. Two top hit genes from the comparison of ZG vs ZF were functionally studied, ANO4 and NEFM. NEFM, encoding neurofilament medium, was the fourth most up-regulated gene in ZG vs ZF, showing 14.8-fold-fold higher expression levels (p=9.16-12) in ZG than ZF. NEFM was also one of the most down-regulated genes in ZF-like vs ZG-like APAs. Immunohistochemistry (IHC) confirmed selective high expression of NEFM in ZG and ZG-like APAs. Silencing NEFM in H295R cells increased aldosterone secretion and cell proliferation. In addition, it increased stimulation and inhibition, respectively, of aldosterone secretion from H295R cells by the dopamine receptor D1R agonist fenoldopam and antagonist SCH23390. IHC showed predominantly intracellular staining for D1R in NEFM-rich ZG-like APAs, but membranous staining in NEFM-poor ZF-like APAs. Aldosterone secretion in response to fenoldopam in primary cells from ZG-like APAs was lower than in cells from ZF-like APAs. NEFM expression levels directly correlate with KCNJ5 phenotype: KCNJ5 mutations down-regulate NEFM mRNA and protein levels in H295R cells and in primary cells from ZG-like APAs. ANO4,encoding a Ca2+-activated chloride channel family member, was the third most upregulated gene, showing 19.9-fold higher expression levels (p=6.6x10-24) in ZG than ZF. IHC confirmed ZG selectivity of ANO4 protein in the adrenal cortex. The staining was mainly cytoplasmic. Unlike NEFM, there was no difference in expression of ANO4 between ZG- and ZF-like APAs, the levels being mid-way between those of ZF and ZG. Overexpression of ANO4 in H295R cells caused an increase in CYP11B2 and NR4A2 gene expression levels but basal aldosterone secretion was unchanged. In the presence of calcium agonists, ANO4 reduced aldosterone secretion. ANO4 subcellular localisation was confirmed as cytoplasmic by immunofluorescence microscopy of transfected cells. When exposed to calcium ionophores, ANO4 generated small chloride currents as detected by YFP assay. In summary, the comparison of transcriptome of ZG with paired ZF found unexpected up-regulated genes. Most of the highly up regulated genes in human ZG, including NEFM and ANO4, inhibit either basal or stimulated aldosterone secretion, and this may reflect an adaptive response to high salt intake. No clear-cut correspondence was found between transcriptome of APAs and their resembling zone of adrenal cortex. The down-regulation of NEFM following transfection of mutant KCNJ5 suggests that ZF-like properties may be a consequence of mutation, rather than tissue of origin.