Modulation of inflammatory mediators by Opuntia ficus-indica and Prunus avium bioproducts using an in vitro cell-based model of intestinal inflammation

Dissertation to obtain a Master Degree in Biotechnology

Stochastic model of transcription initiation of closely spaced promoters in escherichia coli

Dissertação para obtenção do Grau de Mestre em Engenharia Biomédica

Using polymers to improve the rutting resistance of asphalt concrete

This study assesses rutting on two types of modified asphalt mixtures containing: (i) amorphous polyolefin polymer and (ii) a particular polymer obtained by combining LDPE (low density polyethylene) and EVA (ethyl-vinyl-acetate). Rutting tests were performed by a wheel tracking device. Stiffness and fatigue tests were carried out to confirm the performance of the asphalt mixtures. The testing showed that polymer modification in this study improved rut resistance without compromising the stiffness and fatigue behavior. The rutting results were fit in the NCHRP 1-37A model and the in situ rutting performance of asphalt mixtures can be predicted.

Influence of pore size distribution on drying behaviour of porous media by a continuous model

Bundle of capillaries, drying kinetics, continuous model, relative permeability, capillary pressure, control volume method

Sumoylated PPARalpha mediates sex-specific gene repression and protects the liver from estrogen-induced toxicity in mice.

As most metabolic studies are conducted in male animals, understanding the sex specificity of the underlying molecular pathways has been broadly neglected; for example, whether PPARs elicit sex-dependent responses has not been determined. Here we show that in mice, PPARalpha has broad female-dependent repressive actions on hepatic genes involved in steroid metabolism and immunity. In male mice, this effect was reproduced by the administration of a synthetic PPARalpha ligand. Using the steroid oxysterol 7alpha-hydroxylase cytochrome P4507b1 (Cyp7b1) gene as a model, we elucidated the molecular mechanism of this sex-specific PPARalpha-dependent repression. Initial sumoylation of the ligand-binding domain of PPARalpha triggered the interaction of PPARalpha with GA-binding protein alpha (GABPalpha) bound to the target Cyp7b1 promoter. Histone deacetylase and DNA and histone methylases were then recruited, and the adjacent Sp1-binding site and histones were methylated. These events resulted in loss of Sp1-stimulated expression and thus downregulation of Cyp7b1. Physiologically, this repression conferred on female mice protection against estrogen-induced intrahepatic cholestasis, the most common hepatic disease during pregnancy, suggesting a therapeutic target for prevention of this disease.

Relating the permeability of quartz sands to their grain size and spectral induced polarization characteristics

Recently, Revil & Florsch proposed a novel mechanistic model based on the polarization of the Stern layer relating the permeability of granular media to their spectral induced polarization (SIP) characteristics based on the formation of polarized cells around individual grains. To explore the practical validity of this model, we compare it to pertinent laboratory measurements on samples of quartz sands with a wide range of granulometric characteristics. In particular, we measure the hydraulic and SIP characteristics of all samples both in their loose, non-compacted and compacted states, which might allow for the detection of polarization processes that are independent of the grain size. We first verify the underlying grain size/permeability relationship upon which the model of Revil & Florsch is based and then proceed to compare the observed and predicted permeability values for our samples by substituting the grain size characteristics by corresponding SIP parameters, notably the so-called Cole-Cole time constant. In doing so, we also asses the quantitative impact of an observed shift in the Cole-Cole time constant related to textural variations in the samples and observe that changes related to the compaction of the samples are not relevant for the corresponding permeability predictions. We find that the proposed model does indeed provide an adequate prediction of the overall trend of the observed permeability values, but underestimates their actual values by approximately one order-of-magnitude. This discrepancy in turn points to the potential importance of phenomena, which are currently not accounted for in the model and which tend to reduce the characteristic size of the prevailing polarization cells compared to the considered model, such as, for example, membrane polarization, contacts of double-layers of neighbouring grains, and incorrect estimation of the size of the polarized cells because of the irregularity of natural sand grains.

Conditioned polarized Caco-2 cell monolayers allow to discriminate for the ability of gut-derived microorganisms to modulate permeability and antigen-induced basophil degranulation.

BACKGROUND: Food allergy is a common allergic disorder--especially in early childhood. The avoidance of the allergenic food is the only available method to prevent further reactions in sensitized patients. A better understanding of the immunologic mechanisms involved in this reaction would help to develop therapeutic approaches applicable to the prevention of food allergy. OBJECTIVE: To establish a multi-cell in vitro model of sensitized intestinal epithelium that mimics the intestinal epithelial barrier to study the capacity of probiotic microorganisms to modulate permeability, translocation and immunoreactivity of ovalbumin (OVA) used as a model antigen. METHODS: Polarized Caco-2 cell monolayers were conditioned by basolateral basophils and used to examine apical to basolateral transport of OVA by ELISA. Activation of basophils with translocated OVA was measured by beta-hexosaminidase release assay. This experimental setting was used to assess how microorganisms added apically affected these parameters. Basolateral secretion of cytokine/chemokines by polarized Caco-2 cell monolayers was analysed by ELISA. RESULTS: Basophils loaded with OVA-specific IgE responded to OVA in a dose-dependent manner. OVA transported across polarized Caco-2 cell monolayers was found to trigger basolateral basophil activation. Microorganisms including lactobacilli and Escherichia coli increased transepithelial electrical resistance while promoting OVA passage capable to trigger basophil activation. Non-inflammatory levels of IL-8 and thymic stromal lymphopoietin were produced basolaterally by Caco-2 cells exposed to microorganisms. CONCLUSION: The complex model designed in here is adequate to learn about the consequence of the interaction between microorganisms and epithelial cells vis-a-vis the barrier function and antigen translocation, two parameters essential to mucosal homeostasis. It can further serve as a direct tool to search for microorganisms with anti-allergic and anti-inflammatory properties.

Development of a coculture model of encapsulated cells.

In the whole animal, metabolic regulations are set by reciprocal interactions between various organs, via the blood circulation. At present, analyses of such interactions require numerous and uneasily controlled in vivo experiments. In a search for an alternative to in vivo experiments, our work aims at developing a coculture system in which different cell types are isolated in polymer capsules and grown in a common environment. The signals exchanged between cells from various origins are, thus, reproducing the in vivo intertissular communications. With this perspective, we evaluated a new encapsulation system as an artificial housing for liver cells on the one hand and adipocytes on the other hand. Murine hepatocytes were encapsulated with specially designed multicomponent capsules formed by polyelectrolyte complexation between sodium alginate, cellulose sulphate and poly(methylene-coguanidine) hydrochloride, of which the permeability has been characterized. We demonstrated the absence of cytotoxicity and the excellent biocompatibility of these capsules towards primary culture of murine hepatocytes. Encapsulated hepatocytes retain their specific functions--transaminase activity, urea synthesis, and protein secretion--during the first four days of culture in minimum medium. Mature adipocytes, isolated from mouse epidydimal fat, were embedded in alginate beads. Measurement of protein secretion shows an identical profile between free and embedded adipocytes. We finally assessed the properties of encapsulated hepatocytes, cryopreserved over a periods of up to four months. The perspective of using encapsulated cells in coculture are discussed, since this system may represent a promising tool for fundamental research, such as analyses of drug metabolism, intercellular regulations, and metabolic pathways, as well as for the establishment of a tissue bank for storage and supply of murine hepatocytes.

Novel relationship between Vegf expression and retinal pigmented epithelium permeability following light damage in the mouse retina

Purpose:In the retina, the balance between pro- and anti-angiogenic factors is critical for angiogenesis control but is also involved in cell survival and maintenance. For instance, the anti-angiogenic factor PEDF is neuroprotective for photoreceptors (PRs) in models of retinal degeneration. We previously reported upregulation of VEGF (24h to 48h post lesion) in the light-damage (LD) model. Furthermore, systemic delivery of PEDF, as well as lentiviral gene transfer of an anti-VEGF antibody rescue PRs from cell death. Studies in vitro show that VEGF induces retinal endothelial cells apoptosis via the alteration of the Akt1/p38 MAPK signalling pathway under hypoxic conditions. Thus, in this study, we investigate the effect of high levels of VEGF on retinal pigmented epithelium (RPE) permeability and molecular targets expression after light-induced PR degeneration. Methods:To characterize the action of VEGF in the retina during the course of LD, we exposed adult Balb/c mice to 5'000 lux for 1h, and we collected neural retinas and eye-cups (containing RPE) at different time points after the LD. We analysed protein expression by Elisa and Western blotting. In order to study RPE cell permeability after the LD we stained β-catenin on flat mounted RPE. Results:In the neural retina, preliminary results indicate that high levels of VEGF induce a significant upregulation of VEGF receptor 2, whereas VEGF receptor 1 expression is decreased. Concomitantly with VEGF upregulation, LD increases the Src phosphorylation between 24h to 48h. Furthermore, we observe that β-catenin translocates to the cytoplasm of RPE cells between 24h to 36h after the lesion, indicating an increase on the RPE permeability, which could contribute indirectly to the deleterious effect of VEGF observed during light-induced PR apoptosis. Conclusions:This study further involves VEGF in LD and highlights the prime importance of angiogenic factor balance for PR survival. Our results suggest that PR apoptosis is augmented by RPE cell permeability, which may induce high level of VEGF and could be deleterious. The specific action of RPE permeability on PR survival and the role of Src in the retina are under investigation.

An in vitro model for dengue virus infection that exhibits human monocyte infection, multiple cytokine production and dexamethasone immunomodulation

An important cytokine role in dengue fever pathogenesis has been described. These molecules can be associated with haemorrhagic manifestations, coagulation disorders, hypotension and shock, all symptoms implicated in vascular permeability and disease worsening conditions. Several immunological diseases have been treated by cytokine modulation and dexamethasone is utilized clinically to treat pathologies with inflammatory and autoimmune ethiologies. We established an in vitro model with human monocytes infected by dengue virus-2 for evaluating immunomodulatory and antiviral activities of potential pharmaceutical products. Flow cytometry analysis demonstrated significant dengue antigen detection in target cells two days after infection. TNF-alpha, IFN-alpha, IL-6 and IL-10 are produced by in vitro infected monocytes and are significantly detected in cell culture supernatants by multiplex microbead immunoassay. Dexamethasone action was tested for the first time for its modulation in dengue infection, presenting optimistic results in both decreasing cell infection rates and inhibiting TNF-alpha, IFN-alpha and IL-10 production. This model is proposed for novel drug trials yet to be applyed for dengue fever.

The effect of COX-2 inhibitors on the formation of heterotopic bone in a rat model : O1323

Aims: 1) to create a new and reproducible animal model to produce heterotopic ossification (HO) 2) to be able to exactly quantify the amount of HO using a microCT scan and 3) to prove the hypothesis that COX-2 inhibitors are efficacious in the prevention of HO. Methods: We developed a IACUC-approved Lewis rat model, in which the ventral side of the right femur was scraped to mechanically disrupt the periosteum. By clamping the vastus intermedius ischemic injury to the muscle was produced to enhance HO. Finally homologous bone marrow from a donor rat was placed on the anterior surface of the femur. Half of the study group (8 rats) received chow mixed with a COX-2 inhibitor, while the other half received normal chow. After 6 weeks the animals were sacrificed, the femurs removed and imaged by microCT. Grading of HO was based on the thickness of ectopic bone as evaluated in a blinded fashion by 3 independent observers. Results: All animals developed bilateral HO. Rats treated with COX-2 inhibitors developed significantly less ectopic bone than the control group rats. Conclusions: The results suggest that we have created a very reliable, reproducible model to form ectopic bone in rats. Using the microCT we can precisely quantify the amount of HO. We have been able to show that COX-2 inhibitors significantly decrease the amount of HO formation and are thus a good alternative to non-specific NSAIDs with their potential serious side effects on the gastrointestinal tract and on hemo-stastis.

Uncertainty Quantification Of A Semi-Supervised Support Vector Regression Reservoir Model

Uncertainty quantification of petroleum reservoir models is one of the present challenges, which is usually approached with a wide range of geostatistical tools linked with statistical optimisation or/and inference algorithms. Recent advances in machine learning offer a novel approach to model spatial distribution of petrophysical properties in complex reservoirs alternative to geostatistics. The approach is based of semisupervised learning, which handles both ?labelled? observed data and ?unlabelled? data, which have no measured value but describe prior knowledge and other relevant data in forms of manifolds in the input space where the modelled property is continuous. Proposed semi-supervised Support Vector Regression (SVR) model has demonstrated its capability to represent realistic geological features and describe stochastic variability and non-uniqueness of spatial properties. On the other hand, it is able to capture and preserve key spatial dependencies such as connectivity of high permeability geo-bodies, which is often difficult in contemporary petroleum reservoir studies. Semi-supervised SVR as a data driven algorithm is designed to integrate various kind of conditioning information and learn dependences from it. The semi-supervised SVR model is able to balance signal/noise levels and control the prior belief in available data. In this work, stochastic semi-supervised SVR geomodel is integrated into Bayesian framework to quantify uncertainty of reservoir production with multiple models fitted to past dynamic observations (production history). Multiple history matched models are obtained using stochastic sampling and/or MCMC-based inference algorithms, which evaluate posterior probability distribution. Uncertainty of the model is described by posterior probability of the model parameters that represent key geological properties: spatial correlation size, continuity strength, smoothness/variability of spatial property distribution. The developed approach is illustrated with a fluvial reservoir case. The resulting probabilistic production forecasts are described by uncertainty envelopes. The paper compares the performance of the models with different combinations of unknown parameters and discusses sensitivity issues.

The repressor element silencing transcription factor (REST)-mediated transcriptional repression requires the inhibition of Sp1.

The terminal differentiation of neuronal and pancreatic beta-cells requires the specific expression of genes that are targets of an important transcriptional repressor named RE-1 silencing transcription factor (REST). The molecular mechanism by which these REST target genes are expressed only in neuronal and beta-cells and are repressed by REST in other tissues is a central issue in differentiation program of neuronal and beta-cells. Herein, we showed that the transcriptional factor Sp1 was required for expression of most REST target genes both in insulin-secreting cells and neuronal-like cells where REST is absent. Inhibition of REST in a non-beta and a non-neuronal cell model restored the transcriptional activity of Sp1. This activity was also restored by trichostatin A indicating the requirement of histone deacetylases for the REST-mediated silencing of Sp1. Conversely, exogenous introduction of REST blocked Sp1-mediated transcriptional activity. The REST inhibitory effect was mediated through its C-terminal repressor domain, which could interact with Sp1. Taken together, these data show that the inhibition of Sp1 by REST is required for the silencing of its target genes expression in non-neuronal and in non-beta-cells. We conclude that the interplay between REST and Sp1 determines the cell-specific expression of REST target genes.

PlGF-1 and VEGFR-1 pathway regulation of the external epithelial hemato-ocular barrier. A model for retinal edema.

VEGF is considered as an important factor in the pathogenesis of macular edema. VEGF induces the rupture of the blood retinal barrier and may also influence the retinal pigment epithelial (RPE) outer retinal barrier. The aim of this work was to analyze the influence of the VEGF receptor pathways in the modulation of the RPE barrier breakdown in vitro and in vivo. The ARPE19 human junctions in culture are modulated by VEGF through VEGFR-1 but not through VEGFR-2. PlGF-1, that is a pure agonist of VEGFR-1, is produced in ARPE-19 cells under hypoxic conditions and mimics VEGF effects on the external retinal barrier as measured by TER and inulin flux. In vivo, the intravitreous injection of PlGF-1 induces a rupture of the external retinal barrier together with a retinal edema. This effect is reversible within 4 days. VEGF-E, that is a pure agonist of VEGFR-2, does not induce any acute effect on the RPE barrier. These results demonstrate that PlGF-1 can reproduce alterations of the RPE barrier occurring during diabetic retinopathy.

Stent-assisted coiling of bifurcation aneurysms may improve endovascular treatment: a critical evaluation in an experimental model.

BACKGROUND AND PURPOSE: Endovascular treatment of wide-neck bifurcation aneurysms often results in incomplete occlusion or aneurysm recurrence. The goals of this study were to compare results of coil embolization with or without the assistance of self-expandable stents and to examine how stents may influence neointima formation. MATERIALS AND METHODS: Wide-neck bifurcation aneurysms were constructed in 24 animals and, after 4-6 weeks, were randomly allocated to 1 of 5 groups: 1) coil embolization using the assistance of 1 braided stent (n = 5); 2) coil embolization using the assistance of 2 braided stents in a Y configuration (n = 5); 3) coil embolization without stent assistance (n = 6); 4) Y-stenting alone (n = 4); and 5) untreated controls (n = 4). Angiographic results were compared at baseline and at 12 weeks, by using an ordinal scale. Neointima formation at the neck at 12 weeks was compared among groups by using a semiquantitative grading scale. Bench studies were performed to assess stent porosities. RESULTS: Initial angiographic results were improved with single stent-assisted coiling compared with simple coiling (P = .013). Angiographic results at 12 weeks were improved with any stent assistance (P = .014). Neointimal closure of the aneurysm neck was similar with or without stent assistance (P = .908), with neointima covering coil loops but rarely stent struts. Y-stent placement alone had no therapeutic effect. Bench studies showed that porosities can be decreased with stent compaction, but a relatively stable porous transition zone was a limiting factor. CONCLUSIONS: Stent-assisted coiling may improve results of embolization by allowing more complete initial coiling, but these high-porosity stents did not provide a scaffold for more complete neointimal closure of aneurysms.